(14 intermediate revisions by 2 users not shown) | |||
Line 56: | Line 56: | ||
<input type="checkbox" id="Other" name="type" checked="checked" /> <label for="Other">Other</label> | <input type="checkbox" id="Other" name="type" checked="checked" /> <label for="Other">Other</label> | ||
<div class="thirteen wide column" id="labbook-content"> | <div class="thirteen wide column" id="labbook-content"> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
<div class="month" id="April" > | <div class="month" id="April" > | ||
− | + | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
<div class="date">Tuesday 25th</div> | <div class="date">Tuesday 25th</div> | ||
Line 227: | Line 179: | ||
Bacteria lost second plasmid probably. | Bacteria lost second plasmid probably. | ||
</li> | </li> | ||
− | </ul> | + | </ul> |
− | + | ||
− | + | ||
− | + | ||
</div> | </div> | ||
<div class="month" id="May" > | <div class="month" id="May" > | ||
− | |||
<div class="date">Tuesday 2nd</div> | <div class="date">Tuesday 2nd</div> | ||
Line 642: | Line 590: | ||
<img src="https://static.igem.org/mediawiki/2017/a/a5/T--Munich--Results_20170516_Cas13a_Lbu_Heparin_gel.png"> | <img src="https://static.igem.org/mediawiki/2017/a/a5/T--Munich--Results_20170516_Cas13a_Lbu_Heparin_gel.png"> | ||
</li> | </li> | ||
− | </ul> | + | </ul> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<div class="date">Tuesday 23rd</div> | <div class="date">Tuesday 23rd</div> | ||
<ul class="myEntry cas13a"> | <ul class="myEntry cas13a"> | ||
Line 678: | Line 619: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <img src="AGAROSEGEL"> | + | <img src="AGAROSEGEL"> |
</li> | </li> | ||
</li> | </li> | ||
Line 806: | Line 747: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <img src="AGAROSEGEL"> | + | <img src="AGAROSEGEL"> |
</li> | </li> | ||
</li> | </li> | ||
Line 1,378: | Line 1,319: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <img src="GELIMAGE"> | + | <img src="GELIMAGE"> |
</li> | </li> | ||
</ul> | </ul> | ||
Line 1,439: | Line 1,380: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <img src="PCR image (see 8.6.?)"> | + | <img src="PCR image (see 8.6.?)"> |
</li> | </li> | ||
</ul> | </ul> | ||
Line 1,655: | Line 1,596: | ||
</ul> | </ul> | ||
− | + | ||
<div class="date">Friday 16th</div> | <div class="date">Friday 16th</div> | ||
Line 1,706: | Line 1,647: | ||
The Cas13a proteins were activated and cleaved the aptamers. This resulted in a decrease in fluorescence intensity. | The Cas13a proteins were activated and cleaved the aptamers. This resulted in a decrease in fluorescence intensity. | ||
</li> | </li> | ||
− | <img src="A lot of images!!!!"> | + | <img src="A lot of images!!!!"> |
</li> | </li> | ||
</ul> | </ul> | ||
Line 1,928: | Line 1,869: | ||
</ul> | </ul> | ||
− | + | ||
<div class="date">Friday 23rd</div> | <div class="date">Friday 23rd</div> | ||
Line 2,339: | Line 2,280: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <img src="GELIMAGEs.5.7."> | + | <img src="GELIMAGEs.5.7."> |
</li> | </li> | ||
</ul> | </ul> | ||
Line 2,424: | Line 2,365: | ||
</ul> | </ul> | ||
− | + | ||
<div class="date">Friday 7th</div> | <div class="date">Friday 7th</div> | ||
Line 2,449: | Line 2,390: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <img src="GELIMAGE"> | + | <img src="GELIMAGE"> |
</li> | </li> | ||
</ul> | </ul> | ||
Line 2,509: | Line 2,450: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <img src="graph"> | + | <img src="graph"> |
</li> | </li> | ||
</ul> | </ul> | ||
Line 2,526: | Line 2,467: | ||
</li> | </li> | ||
− | </ul | + | </ul> |
<div class="date">Friday 14th</div> | <div class="date">Friday 14th</div> | ||
Line 2,568: | Line 2,509: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <img src="GRAPH"> | + | <img src="GRAPH"> |
</li> | </li> | ||
Line 2,753: | Line 2,694: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <img src="GELIMAGE maybe in next days"> | + | <img src="GELIMAGE maybe in next days"> |
</li> | </li> | ||
Line 2,828: | Line 2,769: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <img src="GELIMAGE"> | + | <img src="GELIMAGE"> |
</li> | </li> | ||
Line 2,881: | Line 2,822: | ||
<div class="month" id="August" > | <div class="month" id="August" > | ||
<div class="date firstOfMonth">Tuesday 1st</div> | <div class="date firstOfMonth">Tuesday 1st</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">DNA Gel-Purification and Genomic DNA Purification</a | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">DNA Gel-Purification and Genomic DNA Purification</a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 2,898: | Line 2,839: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Purification of b-Gal fragments from 1 % Agarose Gel <!-- | + | Purification of b-Gal fragments from 1 % Agarose Gel <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 2,908: | Line 2,849: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
DNA was lost during the purification | DNA was lost during the purification | ||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Wednesday 2nd</div> | <div class="date">Wednesday 2nd</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Q5-PCR and PCR-Cleanup kit</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Q5-PCR and PCR-Cleanup kit</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 2,930: | Line 2,871: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Repeated PCR of beta-Gal N and C Terminal fragments from 31.7. <!-- | + | Repeated PCR of beta-Gal N and C Terminal fragments from 31.7. <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 2,988: | Line 2,929: | ||
</ul> | </ul> | ||
<div class="date">Thursday 3rd</div> | <div class="date">Thursday 3rd</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Agarose-Gel</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Agarose-Gel</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 3,005: | Line 2,946: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Gel of purified PCR <!-- | + | Gel of purified PCR <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,015: | Line 2,956: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
<img src="https://static.igem.org/mediawiki/2017/c/c4/T--Munich--Results_20170803_Max_Gel.png"> | <img src="https://static.igem.org/mediawiki/2017/c/c4/T--Munich--Results_20170803_Max_Gel.png"> | ||
<ul> | <ul> | ||
Line 3,022: | Line 2,963: | ||
</li> | </li> | ||
</ul> | </ul> | ||
− | + | ||
<div class="date">Monday 7th</div> | <div class="date">Monday 7th</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Protein-Purifcation of Lbu and Lsh</b> <!-- | + | <b>Protein-Purifcation of Lbu and Lsh</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols"> - </a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols"> - </a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max, Ludwig, Teeradon <!-- | + | Participants: Max, Ludwig, Teeradon <!-- --> |
</li> | </li> | ||
Line 3,042: | Line 2,983: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Inoculation of main cultures of Rosetta p2CT-His-MBP-Lbu_C13a_WT and Lsh in LB(Carb+Cm) <!-- | + | Inoculation of main cultures of Rosetta p2CT-His-MBP-Lbu_C13a_WT and Lsh in LB(Carb+Cm) <!--All--> |
</li> | </li> | ||
Line 3,074: | Line 3,015: | ||
</ul> | </ul> | ||
<div class="date">Tuesday 8th</div> | <div class="date">Tuesday 8th</div> | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>First test with 3D printed 96-well plate</b> <!-- | + | <b>First test with 3D printed 96-well plate</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 3,091: | Line 3,032: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Tested different concentrations of fluorescein in a 3D printed 96 well plate on paper. <!-- | + | Tested different concentrations of fluorescein in a 3D printed 96 well plate on paper. <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,103: | Line 3,044: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
No light bleeding was observed in the experiment | No light bleeding was observed in the experiment | ||
</li> | </li> | ||
Line 3,136: | Line 3,077: | ||
<div class="date">Wednesday 9th</div> | <div class="date">Wednesday 9th</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Expression and Harvesting of Cas13a variants</b> <!-- | + | <b>Expression and Harvesting of Cas13a variants</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Protein Purification</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Protein Purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max, Ludwig <!-- | + | Participants: Max, Ludwig <!-- --> |
</li> | </li> | ||
Line 3,153: | Line 3,094: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Harvested Cas13a Lbu overexpression. <!-- | + | Harvested Cas13a Lbu overexpression. <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,198: | Line 3,139: | ||
<div class="date">Thursday 10th</div> | <div class="date">Thursday 10th</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>His Purification of Cas13a Lbu and Harvesting of Cas13a Lsh</b> <!-- | + | <b>His Purification of Cas13a Lbu and Harvesting of Cas13a Lsh</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His-Purification</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His-Purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max, Ludwig, Milica, Sven, Benedikt <!-- | + | Participants: Max, Ludwig, Milica, Sven, Benedikt <!-- --> |
</li> | </li> | ||
Line 3,294: | Line 3,235: | ||
</ul> | </ul> | ||
<div class="date">Friday 11th</div> | <div class="date">Friday 11th</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Reverse HIS purification and SEC of Cas13a Lbu</b> <!-- | + | <b>Reverse HIS purification and SEC of Cas13a Lbu</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His purification and SEC protein purification</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His purification and SEC protein purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Ludwig, Benedikt, Max, Sven <!-- | + | Participants: Ludwig, Benedikt, Max, Sven <!-- --> |
</li> | </li> | ||
Line 3,311: | Line 3,252: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Incubation of cleaved Lbu sample with Ni-NTA beads (ca. 7 mL) (1 h, 4 °C) <!-- | + | Incubation of cleaved Lbu sample with Ni-NTA beads (ca. 7 mL) (1 h, 4 °C) <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,330: | Line 3,271: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
See Gel image of 14.8. for results. | See Gel image of 14.8. for results. | ||
</li> | </li> | ||
Line 3,397: | Line 3,338: | ||
</ul> | </ul> | ||
<div class="date">Monday 14th</div> | <div class="date">Monday 14th</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>SDS PAGE of Lbu purifcations</b> <!-- | + | <b>SDS PAGE of Lbu purifcations</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">SDS-PAGE</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">SDS-PAGE</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Ludwig <!-- | + | Participants: Ludwig <!-- --> |
</li> | </li> | ||
Line 3,414: | Line 3,355: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | SDS-PAGE of Lbu purification from last week <!-- | + | SDS-PAGE of Lbu purification from last week <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,478: | Line 3,419: | ||
</ul> | </ul> | ||
<div class="date">Wednesday 16th</div> | <div class="date">Wednesday 16th</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Cas13a preparation</b> <!-- | + | <b>Cas13a preparation</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Benedikt, Ludwig <!-- | + | Participants: Benedikt, Ludwig <!-- --> |
</li> | </li> | ||
Line 3,495: | Line 3,436: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Concentration measurment of Lbu, Lsh and Lwa with Bradford essay <!-- | + | Concentration measurment of Lbu, Lsh and Lwa with Bradford essay <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,507: | Line 3,448: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
Concentration of proteins measured(µg/ml): | Concentration of proteins measured(µg/ml): | ||
<li> | <li> | ||
Line 3,560: | Line 3,501: | ||
</li> | </li> | ||
</ul> | </ul> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Protein-Expression of Lbu and Lwa</b> <!-- | + | <b>Protein-Expression of Lbu and Lwa</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Protein-Expression</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Protein-Expression</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Benedikt, Ludwig, Milica <!-- | + | Participants: Benedikt, Ludwig, Milica <!-- --> |
</li> | </li> | ||
Line 3,577: | Line 3,518: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Expression of Rosetta Cas13a Lbu (3L 2xYT Medium, 16°C ON) <!-- | + | Expression of Rosetta Cas13a Lbu (3L 2xYT Medium, 16°C ON) <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,586: | Line 3,527: | ||
</ul> | </ul> | ||
<div class="date">Friday 18th</div> | <div class="date">Friday 18th</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Harvesting and Purification of Cas13a Lbu and Lsh</b> <!-- | + | <b>Harvesting and Purification of Cas13a Lbu and Lsh</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Protein Purification</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Protein Purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Benedikt, Milica <!-- | + | Participants: Benedikt, Milica <!-- --> |
</li> | </li> | ||
Line 3,603: | Line 3,544: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Harvesting of both expressions <!-- | + | Harvesting of both expressions <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,615: | Line 3,556: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
</li> | </li> | ||
Line 3,640: | Line 3,581: | ||
</ul> | </ul> | ||
<div class="date">Monday 21st</div> | <div class="date">Monday 21st</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Continuation of Cas13a purification</b> <!-- | + | <b>Continuation of Cas13a purification</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His-Purification</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His-Purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Benedikt, Milica, Max <!-- | + | Participants: Benedikt, Milica, Max <!-- --> |
</li> | </li> | ||
Line 3,657: | Line 3,598: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Continued His purification of Cas13a Lbu <!-- | + | Continued His purification of Cas13a Lbu <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,671: | Line 3,612: | ||
<ul> | <ul> | ||
<li>As visible on the gel the elutions 1 and 2 contained the highest amount of protein and were pooled for the TEV cleavage.</li> | <li>As visible on the gel the elutions 1 and 2 contained the highest amount of protein and were pooled for the TEV cleavage.</li> | ||
− | </ul> <!-- | + | </ul> <!----> |
</li> | </li> | ||
Line 3,756: | Line 3,697: | ||
</ul> | </ul> | ||
<div class="date">Tuesday 22nd</div> | <div class="date">Tuesday 22nd</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Reverse His purification of Cas13a Lbu</b> <!-- | + | <b>Reverse His purification of Cas13a Lbu</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His purification</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">His purification</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max, Benedikt, Milica <!-- | + | Participants: Max, Benedikt, Milica <!-- --> |
</li> | </li> | ||
Line 3,773: | Line 3,714: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Incubation (shaker!!) of cleaved Lbu sample with Ni-NTA beads (ca. 7 mL) (1 h, ice) <!-- | + | Incubation (shaker!!) of cleaved Lbu sample with Ni-NTA beads (ca. 7 mL) (1 h, ice) <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,792: | Line 3,733: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
</li> | </li> | ||
Line 3,880: | Line 3,821: | ||
</ul> | </ul> | ||
<div class="date">Wednesday 23rd</div> | <div class="date">Wednesday 23rd</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Cas13a purification</b> <!-- | + | <b>Cas13a purification</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Size Exclusion Chromatography</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Size Exclusion Chromatography</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Benedikt, Milica <!-- | + | Participants: Benedikt, Milica <!-- --> |
</li> | </li> | ||
Line 3,897: | Line 3,838: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Measuring concentration after Reverse His Lbu <!-- | + | Measuring concentration after Reverse His Lbu <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 3,909: | Line 3,850: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
See SDS PAGE of 24.8. to see the SEC result. | See SDS PAGE of 24.8. to see the SEC result. | ||
</li> | </li> | ||
Line 3,976: | Line 3,917: | ||
</ul> | </ul> | ||
<div class="date">Thursday 24th</div> | <div class="date">Thursday 24th</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>SDS-Gel of SEC-Run from 23.8.</b> <!-- | + | <b>SDS-Gel of SEC-Run from 23.8.</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">SDS-PAGE</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">SDS-PAGE</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Chris <!-- | + | Participants: Chris <!-- --> |
</li> | </li> | ||
Line 3,993: | Line 3,934: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | 10% SDS PAGE of SEC Run samples <!-- | + | 10% SDS PAGE of SEC Run samples <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 4,003: | Line 3,944: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
<img src="https://static.igem.org/mediawiki/2017/3/31/T--Munich--Results_20170824_Max_Gel.png"> | <img src="https://static.igem.org/mediawiki/2017/3/31/T--Munich--Results_20170824_Max_Gel.png"> | ||
<ul><li>The samples with the highest protein concentration were pooled and used for further experiments.</li></ul> | <ul><li>The samples with the highest protein concentration were pooled and used for further experiments.</li></ul> | ||
Line 4,255: | Line 4,196: | ||
<div class="date">Tuesday 29th</div> | <div class="date">Tuesday 29th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR with Q5 Polymerase, PCR Cleanup Kit, Restriction digest and Ligation</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR with Q5 Polymerase, PCR Cleanup Kit, Restriction digest and Ligation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 4,275: | Line 4,216: | ||
</li> | </li> | ||
<li> | <li> | ||
− | PCR of SPlit Uni C und Split Uni N with Q5 Polymerase <!-- | + | PCR of SPlit Uni C und Split Uni N with Q5 Polymerase <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 4,460: | Line 4,401: | ||
</ul> | </ul> | ||
<div class="date">Thursday 31st</div> | <div class="date">Thursday 31st</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">E. comp Transformation</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">E. comp Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 4,477: | Line 4,418: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | E. comp transformation of E. coli Turbo with 1 µl of overnight ligation of 27.8. <!-- | + | E. comp transformation of E. coli Turbo with 1 µl of overnight ligation of 27.8. <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 4,621: | Line 4,562: | ||
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: | + | Protocol: |
<img src="https://static.igem.org/mediawiki/2017/9/91/T--Munich--pic--rnaextraction_pipettingScheme.png" > | <img src="https://static.igem.org/mediawiki/2017/9/91/T--Munich--pic--rnaextraction_pipettingScheme.png" > | ||
</li> | </li> | ||
Line 4,663: | Line 4,604: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Three lysis methods were used: sonifcation, lysozyme digestion and heat lysis in SDS. | + | Three lysis methods were used: sonifcation, lysozyme digestion and heat lysis in SDS. All |
</li> | </li> | ||
<li> | <li> | ||
Line 4,686: | Line 4,627: | ||
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: < | + | Protocol: |
+ | <img src="https://static.igem.org/mediawiki/2017/9/91/T--Munich--pic--rnaextraction_pipettingScheme.png" > | ||
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
Line 4,711: | Line 4,653: | ||
<div class="date">Monday 4th</div> | <div class="date">Monday 4th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">E. comp Transformation</a | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">E. comp Transformation</a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 4,728: | Line 4,670: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Plates showed no colonies as there was no SOC step. <!-- | + | Plates showed no colonies as there was no SOC step. <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 4,789: | Line 4,731: | ||
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: < | + | Protocol: |
+ | <img src="https://static.igem.org/mediawiki/2017/9/91/T--Munich--pic--rnaextraction_pipettingScheme.png" > | ||
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
Line 4,815: | Line 4,758: | ||
</ul> | </ul> | ||
<div class="date">Tuesday 5th</div> | <div class="date">Tuesday 5th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry other"> |
+ | <li class="myTitel"> | ||
+ | <b>DNA Amplification Test using Recombinase Polymerase Amplification </b> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Recombinase Polymerase Amplification</a> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Sven | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | DNA Amplification with RPA was tested using the provided Control Reaction by TwistDx and our BioBrick His6-TEV plasmid | ||
+ | incubation times of 40 minutes for Control and Sample 1 and 60 minutes for sample 2. Differences due to incubation time | ||
+ | was tested. | ||
+ | </li> | ||
+ | <li> | ||
+ | RPA according to protocol | ||
+ | </li> | ||
+ | <li> | ||
+ | PCR cleanup according to protocol. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | |||
+ | <ul> | ||
+ | <li>Positive Control worked.</li> | ||
+ | <li>RPA of TEV could not produce the whole (~1300 bp) construct but would produce amplicons of shorter size (100-500 bp) which was anticipated since TwistDx protocol says optimal amplicon length is 500 bp.</li> | ||
+ | <li>Full length double-stranded DNA could consequently not be formed.</li> | ||
+ | </ul> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/24/T--Munich--Results_20170905_test_RPA_Cy2.png"> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Agarose-Gel</a | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Agarose-Gel</a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 4,832: | Line 4,815: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Ran a small 1.75% TAE-Agarose Gel at 150 V for 50 minutes and post stained with Sybr Gold <!-- | + | Ran a small 1.75% TAE-Agarose Gel at 150 V for 50 minutes and post stained with Sybr Gold <!--All--> |
</li> | </li> | ||
</ul> | </ul> | ||
Line 4,838: | Line 4,821: | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Overnight Ligation of Intein-Extein-Construct</b> <!-- | + | <b>Overnight Ligation of Intein-Extein-Construct</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Ligation of DNA-Fragments</a | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Ligation of DNA-Fragments</a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 4,853: | Line 4,836: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Overnight ligation in ice bath with psb1C3 and psb4A5 as backbones and a 3:1 molar excess of insert <!-- | + | Overnight ligation in ice bath with psb1C3 and psb4A5 as backbones and a 3:1 molar excess of insert <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 4,863: | Line 4,846: | ||
</ul> | </ul> | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Preparation of chemical competent Turbo cells</b> <!-- | + | <b>Preparation of chemical competent Turbo cells</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Preparation of chemical competent cells</a | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Preparation of chemical competent cells</a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 4,880: | Line 4,863: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Plated one aliquot as negative controll <!-- | + | Plated one aliquot as negative controll <!--All--> |
</li> | </li> | ||
Line 4,887: | Line 4,870: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | No colonies on plates, therefore usefull competent cells. <!-- | + | No colonies on plates, therefore usefull competent cells. <!----> |
</li> | </li> | ||
Line 4,983: | Line 4,966: | ||
<div class="date">Wednesday 6th</div> | <div class="date">Wednesday 6th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry cas13a"> |
+ | <li class="myTitel"> | ||
+ | <b> RNaseAlert Experiment with E.coli RNA with different concentrations of target E.coli RNA </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa, Jorge, Igor | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Run1: Amount of RNA: 1 nm Cas13a, 10 nm crRNA | ||
+ | </li> | ||
+ | <li> | ||
+ | Run2: Amount of RNA: 1 nm Cas13a, 10 nm crRNA | ||
+ | </li> | ||
+ | <li> | ||
+ | img src="https://static.igem.org/mediawiki/2017/b/be/T--Munich--pic--platereader_pipScheme_6_09_Ecoli.png" > | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | |||
+ | </ul> | ||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Chem and electro Transformation</a | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Chem and electro Transformation</a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,000: | Line 5,009: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Transformation into chem and e. competent E.coli Turbo and DH5 alpha cells. <!-- | + | Transformation into chem and e. competent E.coli Turbo and DH5 alpha cells. <!--All--> |
</li> | </li> | ||
Line 5,007: | Line 5,016: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | No colonies in the evening, therefore the plates were left at room temperature overnight. <!-- | + | No colonies in the evening, therefore the plates were left at room temperature overnight. <!----> |
</li> | </li> | ||
Line 5,036: | Line 5,045: | ||
</ul> | </ul> | ||
<div class="date">Thursday 7th</div> | <div class="date">Thursday 7th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>RNA Intein-Extein-Readout</b> <!-- | + | <b>RNA Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Colony PCR</a | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Colony PCR</a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,053: | Line 5,062: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Picked 128 colonies from plates of 06.09.17 <!-- | + | Picked 128 colonies from plates of 06.09.17 <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,132: | Line 5,141: | ||
<div class="date">Monday 11th</div> | <div class="date">Monday 11th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols"> - </a | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols"> - </a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,149: | Line 5,158: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Precultures of 16 colonies from plates of 06.09.17. <!-- | + | Precultures of 16 colonies from plates of 06.09.17. <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,182: | Line 5,191: | ||
<div class="date">Tuesday 12th</div> | <div class="date">Tuesday 12th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Mini-Prep with Qiagen Kit and Sequencing Sample preparation </a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Mini-Prep with Qiagen Kit and Sequencing Sample preparation </a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,199: | Line 5,208: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Mini Prep of precultures from 11.09.17 <!-- | + | Mini Prep of precultures from 11.09.17 <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,209: | Line 5,218: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | psB4A5_C-Terminal 1-7 and 3-4 were positive.<!-- | + | psB4A5_C-Terminal 1-7 and 3-4 were positive.<!----> |
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Wednesday 13th</div> | <div class="date">Wednesday 13th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest and PCR protocol with Q5 polymerase</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest and PCR protocol with Q5 polymerase</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,231: | Line 5,240: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Send Samples psB4A5_C-Terminal 1-7 and 3-4 for sequencing with primer VF <!-- | + | Send Samples psB4A5_C-Terminal 1-7 and 3-4 for sequencing with primer VF <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,243: | Line 5,252: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
PsB4A5_C terminal 1-7 was sequenced completly and used for further cloning steps. | PsB4A5_C terminal 1-7 was sequenced completly and used for further cloning steps. | ||
</li> | </li> | ||
Line 5,305: | Line 5,314: | ||
</ul> | </ul> | ||
<div class="date">Thursday 14th</div> | <div class="date">Thursday 14th</div> | ||
− | <ul class="myEntry | + | <ul class="myEntry other"> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b> | + | <b>RPA stability test and Reaction Temperature Screening on paper </b> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols"> | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Recombinase Polymerase Amplification on Paper</a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: | + | Participants: Sven |
</li> | </li> | ||
Line 5,322: | Line 5,331: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | New digestion of unamplified N-Terminal gBlock and psB1C3 with SpeI-HF and EcoRI-Hf <!-- | + | Preparation of stability assay of RPA on paper. Lyophilisation and storage. |
+ | </li> | ||
+ | <li> | ||
+ | RPA on paper according to protocol | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | |||
+ | </ul> | ||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
+ | <li class="myTitel"> | ||
+ | <b>Intein-Extein-Readout</b> <!----> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest, Ligation of DNA fragments, Gibbson-Assembly and E and chem Trafo</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Max <!-- --> | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | New digestion of unamplified N-Terminal gBlock and psB1C3 with SpeI-HF and EcoRI-Hf <!--All--> | ||
</li> | </li> | ||
<li> | <li> | ||
Line 5,341: | Line 5,376: | ||
</ul> | </ul> | ||
<div class="date">Friday 15th</div> | <div class="date">Friday 15th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry other"> |
+ | <li class="myTitel"> | ||
+ | <b>RPA stability test and Reaction Temperature Screening on paper </b> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Recombinase Polymerase Amplification on Paper</a> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Sven | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Stability of RPA on paper was tested using His6-TEV plasmid after 0, 2 and 24 hours post-lyophilisation. Reaction temperature was tested at 37 °C | ||
+ | as suggested by TwistDx and room temperature. Reaction was positively controlled using non-lyophilised reaction mixture. | ||
+ | </li> | ||
+ | <li> | ||
+ | RPA according to protocol | ||
+ | </li> | ||
+ | <li> | ||
+ | PCR cleanup according to protocol. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | <ul> | ||
+ | <li>Lyophilisation of RPA on paper worked.</li> | ||
+ | <li>Directly after lyophilisation, the reaction | ||
+ | efficiency seems to be similar to fresh RPA reaction mixture.</li> | ||
+ | <li>Lyophilisation of RPA on paper worked. However, activity loss is observed already after 2 hours and after 24 hours, | ||
+ | almost no activity is observable anymore. </li> | ||
+ | </ul> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/1/12/T--Munich--Results_20170915_RPA_Stability_1.png"> | ||
+ | |||
+ | </li> | ||
+ | </ul> | ||
+ | |||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Colony PCR</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Colony PCR</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,358: | Line 5,435: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Picked 32 colonies from plates of 14.09.17 <!-- | + | Picked 32 colonies from plates of 14.09.17 <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,370: | Line 5,447: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
<img src="https://static.igem.org/mediawiki/2017/5/54/T--Munich--Results_20170915_Max_Gel.png"> | <img src="https://static.igem.org/mediawiki/2017/5/54/T--Munich--Results_20170915_Max_Gel.png"> | ||
<ul><li>No positive colononies in the Col PCR.</li></ul> | <ul><li>No positive colononies in the Col PCR.</li></ul> | ||
Line 5,398: | Line 5,475: | ||
</li> | </li> | ||
</ul> | </ul> | ||
+ | <div class="date">Monday 18th</div> | ||
<ul class="myEntry other"> | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>RPA stability test | + | <b>RPA stability test on paper </b> |
</li> | </li> | ||
Line 5,415: | Line 5,493: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | + | Preparation of temperature and air-sensitivity assay of RPA on paper. | |
− | + | ||
</li> | </li> | ||
<li> | <li> | ||
− | RPA | + | RPA on paper according to protocol. |
− | + | ||
− | + | ||
− | + | ||
</li> | </li> | ||
</ul> | </ul> | ||
Line 5,428: | Line 5,502: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | + | <ul> | |
− | + | <li>Lyophilisation of RPA on paper.</li> | |
− | + | <li>Storage with and without Parafilm enclosure at 4 °C or room temperature.</li> | |
− | + | </ul> | |
− | + | ||
− | + | ||
</li> | </li> | ||
</ul> | </ul> | ||
− | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | |
− | <ul class="myEntry readout"> <!-- | + | |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Q5-PCR and Restriction digestion and Col-PCR and e and chem comp Transformation</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Q5-PCR and Restriction digestion and Col-PCR and e and chem comp Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,454: | Line 5,525: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | New PCR of backbone with psB1A3-fw and psB1A3-rev. Sample was subsequently DpnI digested and purified <!-- | + | New PCR of backbone with psB1A3-fw and psB1A3-rev. Sample was subsequently DpnI digested and purified <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,467: | Line 5,538: | ||
</ul> | </ul> | ||
<div class="date">Tuesday 19th</div> | <div class="date">Tuesday 19th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry other"> |
+ | <li class="myTitel"> | ||
+ | <b> RNA extraction from 5ml overnight culture of B. subtilis </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">Phenol-chlorophorm extraction RNA</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | used heat lysis at 90 °C for 15 mins | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b>Coupling <i>In-Vitro Transcription</i> to RPA </b> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Coupling RPA to In-Vitro Transcription</a> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Sven | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Transcription efficiency when coupled to RPA was determined using a reaction without T7 RNA Polymerase as negative control. | ||
+ | The construct as template used was His6-TEV plasmid. | ||
+ | |||
+ | </li> | ||
+ | <li> | ||
+ | RPA-Tx according to protocol. | ||
+ | </li> | ||
+ | <li> | ||
+ | Phenol-Chloroform-Extraction according to protocol. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | <ul> | ||
+ | <li>In-Vitro Transcription could not be shown.</li> | ||
+ | <li>RNA was detected in the samples in which no T7 RNA Polymerase was added while there are no | ||
+ | bands in the samples that had T7 RNA Polymerase in them. Though unlikely, a labelling error might have occured. </li> | ||
+ | </ul> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/e6/T--Munich--Results_20170919_rna_gelly.png"> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b>RPA stability test on paper </b> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Recombinase Polymerase Amplification on Paper</a> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Sven | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Stability of RPA on paper was tested using His6-TEV plasmid after 24 hours post-lyophilisation. Storage conditions were altered. | ||
+ | Storage at room temperature and at 4 °C were tested. Also, duplicates were done at these temperatures, one sample being always enclosed | ||
+ | with Parafilm to avoid air exposure. | ||
+ | |||
+ | </li> | ||
+ | <li> | ||
+ | RPA on Paper according to protocol | ||
+ | </li> | ||
+ | <li> | ||
+ | PCR cleanup according to protocol. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | <ul> | ||
+ | <li>RPA activity clearly showed that storage temperature did not have a significant effect on the integrity of the RPA reaction mixture.</li> | ||
+ | <li>Exposure to air, however, was identified as the bottleneck for RPA stability. The RPA samples that were protected from exposure to air showed significantly higher</li> | ||
+ | </ul> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2017/2/21/T--Munich--PagePictures_RPA_gel2.png"> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout and Biobrick Recloning</b> <!-- | + | <b>Intein-Extein-Readout and Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Gibbson-Assembly and Ligation</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Gibbson-Assembly and Ligation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max, Rob <!-- | + | Participants: Max, Rob <!-- --> |
</li> | </li> | ||
Line 5,484: | Line 5,653: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Gel of Col-PCR from 18.09.17 <!-- | + | Gel of Col-PCR from 18.09.17 <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,496: | Line 5,665: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
<img src="https://static.igem.org/mediawiki/2017/4/43/T--Munich--Results_20170919_Max_Gel.png"> | <img src="https://static.igem.org/mediawiki/2017/4/43/T--Munich--Results_20170919_Max_Gel.png"> | ||
<ul><li>Sample 1-3 and 2-1 had the correct length, and therefore precultures of these colonies were started overnight.</li></ul> | <ul><li>Sample 1-3 and 2-1 had the correct length, and therefore precultures of these colonies were started overnight.</li></ul> | ||
Line 5,579: | Line 5,748: | ||
</ul> | </ul> | ||
<div class="date">Wednesday 20th</div> | <div class="date">Wednesday 20th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry other"> |
+ | <li class="myTitel"> | ||
+ | <b> Gel analysis of B.subtilis RNA samples </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">Denaturating Urea PAGE</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | 8% gel | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | B. subtilis-PCE 190 ng/ul, B.subtilis heat/lysis-378 ng/ul | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Miniprep with Qiagen Kit</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Miniprep with Qiagen Kit</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,596: | Line 5,788: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Minipreped overnight cultures from 1-3 and 2-1. <!-- | + | Minipreped overnight cultures from 1-3 and 2-1. <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,606: | Line 5,798: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | N-Terminal sample 1-3 was positive. <!-- | + | N-Terminal sample 1-3 was positive. <!----> |
</li> | </li> | ||
</ul> | </ul> | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Col PCR and preparation of e comp. cells</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Col PCR and preparation of e comp. cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,627: | Line 5,819: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Colony PCR of 36 colonies from each of the 3 constructs His-Sumo-Lwa (Lwa(l)), Lwa(lwa(k) and His-Sumo-Lbu each in psB1C3. <!-- | + | Colony PCR of 36 colonies from each of the 3 constructs His-Sumo-Lwa (Lwa(l)), Lwa(lwa(k) and His-Sumo-Lbu each in psB1C3. <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,667: | Line 5,859: | ||
</ul> | </ul> | ||
<div class="date">Thurdsay 21st</div> | <div class="date">Thurdsay 21st</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest, Gibbson-Assembly and Transformation</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest, Gibbson-Assembly and Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,684: | Line 5,876: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Digestion of 1 µg of each backbone (psb1c3_N-term_uni and psb4A5_C-Term_uni with BBS1-HF to open them for Gibbson-Assembly <!-- | + | Digestion of 1 µg of each backbone (psb1c3_N-term_uni and psb4A5_C-Term_uni with BBS1-HF to open them for Gibbson-Assembly <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,707: | Line 5,899: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
</li> | </li> | ||
</ul> | </ul> | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Col-PCR and Sample Sequencing</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Col-PCR and Sample Sequencing</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,728: | Line 5,920: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Send samples for sequencing <!-- | + | Send samples for sequencing <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,737: | Line 5,929: | ||
</ul> | </ul> | ||
− | <div class="date"> | + | <div class="date">Wednesday 20th</div> |
− | + | <ul class="myEntry cs13a"> | |
+ | <li class="myTitel"> | ||
+ | <b> RNase Alert Experiment with Bacillus subtilis using varying concentrations of target RNA </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Pippeting scheme: </br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/c/c4/T--Munich--pic--platereader_pipScheme_20_09_bazillus.png"> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <div class="date">Thursday 21th</div> | ||
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b> B. subtilis RNA extraction using lysozyme </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">Phenol-chlorophorm RNA extraction</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | </ul> | ||
<div class="date">Monday 25th</div> | <div class="date">Monday 25th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry other"> |
+ | <li class="myTitel"> | ||
+ | <b> crRNA in vitro transcription (for B.subtilis, Norovirus. Q5 beta, Hepatitis) | ||
+ | </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">In vitro Transcription</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest, PCR Cleanup Kit, Gibbson Assembly and Transformation</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest, PCR Cleanup Kit, Gibbson Assembly and Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,758: | Line 5,993: | ||
<li> | <li> | ||
Redigest of 1 µg of each backbone with BBS1 and dephoshorilation according to protocol | Redigest of 1 µg of each backbone with BBS1 and dephoshorilation according to protocol | ||
− | <!-- | + | <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,781: | Line 6,016: | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Total</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Total</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,798: | Line 6,033: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | COlPCR and Gel of samples from trafo of Friday 22.09.17 => 30 samples (all non green) psB1C3_LBU <!-- | + | COlPCR and Gel of samples from trafo of Friday 22.09.17 => 30 samples (all non green) psB1C3_LBU <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,808: | Line 6,043: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
All negative. | All negative. | ||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Tuesday 26th</div> | <div class="date">Tuesday 26th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry other"> |
+ | <li class="myTitel"> | ||
+ | <b> crRNA quantification, Urea gel run </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">Denaturating Urea Page</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa, Erika | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | 2 samples each from B. subtilis, Norovirus, Q5 beta and HCV(first diluted 1:5 and second undiluted) | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | |||
+ | </ul> | ||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">ColPCR, Agarosegel</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">ColPCR, Agarosegel</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max, Milica <!-- | + | Participants: Max, Milica <!-- --> |
</li> | </li> | ||
Line 5,830: | Line 6,085: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | ColPCR of 12 colonies from N-term plate from 25.9.17 <!-- | + | ColPCR of 12 colonies from N-term plate from 25.9.17 <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,839: | Line 6,094: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
Samples 1-8 and 2-1 of the N-Terminus looked good => preculture for sequencing. | Samples 1-8 and 2-1 of the N-Terminus looked good => preculture for sequencing. | ||
</li> | </li> | ||
Line 5,845: | Line 6,100: | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Total</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Total</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,862: | Line 6,117: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | COlPCR and Gel of samples from trafo of Friday 22.09.17 => 30 samples (all non green) psB1C3_Lwa_kurz <!-- | + | COlPCR and Gel of samples from trafo of Friday 22.09.17 => 30 samples (all non green) psB1C3_Lwa_kurz <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,871: | Line 6,126: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
All negative. | All negative. | ||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Wednesday 27th</div> | <div class="date">Wednesday 27th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Minipreparation and Sample Sequencing</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Minipreparation and Sample Sequencing</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,893: | Line 6,148: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Miniprep of psB1C3_Split_N_term_bGal 1-8 and 2-1 <!-- | + | Miniprep of psB1C3_Split_N_term_bGal 1-8 and 2-1 <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 5,902: | Line 6,157: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
Both sequences positive. | Both sequences positive. | ||
</li> | </li> | ||
Line 5,930: | Line 6,185: | ||
</ul> | </ul> | ||
<div class="date">Thursday 28th</div> | <div class="date">Thursday 28th</div> | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry other"> |
+ | <li class="myTitel"> | ||
+ | <b> Agarose and Urea gel run with RNA </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">PAGE, Urea PAGE</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa, Erika | ||
+ | </li> | ||
+ | |||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | Agarose Gel didn’t work – The samples seems to be degraded in agarose gel , may be due to contamination of the gel chamber and other materials with Rnase. | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">ColPCR, ON culture</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">ColPCR, ON culture</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 5,947: | Line 6,218: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | ColPCR from samples of 22.09.17 <!-- | + | ColPCR from samples of 22.09.17 <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 6,017: | Line 6,288: | ||
</ul> | </ul> | ||
<div class="date">Friday 29th</div> | <div class="date">Friday 29th</div> | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry cas13a"> |
+ | <li class="myTitel"> | ||
+ | <b> RNaseAlert experiment with extracted RNA targets from E.coli and in vitro transcribed RNAs. </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa, Erika | ||
+ | </li> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Pipetting scheme: </br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/e5/T--Munich--pic--platereader_pipScheme_29_09_ecoli.png"> | ||
+ | </li> | ||
+ | |||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | The results for E.coli look good. Excel table saved in lrz as: 2017_09_29_rnase alert_ecolitarget_invitrotarget.xlsx | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Mini-Prepatation</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Mini-Prepatation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,037: | Line 6,332: | ||
</li> | </li> | ||
<li> | <li> | ||
− | Lwa short send for sequencing with VF and VR <!-- | + | Lwa short send for sequencing with VF and VR <!--All--> |
</li> | </li> | ||
Line 6,044: | Line 6,339: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
Lwa long positive. | Lwa long positive. | ||
</li> | </li> | ||
Line 6,170: | Line 6,465: | ||
<div class="month" id="October" > | <div class="month" id="October" > | ||
<div class="date fistOfMonth">Monday 2nd</div> | <div class="date fistOfMonth">Monday 2nd</div> | ||
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b> Phenol chloroform purification of crRNAs of B. subtilis, NoroV, HCV and Q5 beta | ||
+ | </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">Phenol-chloroform extraction RNA</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
Line 6,197: | Line 6,504: | ||
</ul> | </ul> | ||
<div class="date">Tuesday 3rd</div> | <div class="date">Tuesday 3rd</div> | ||
+ | <ul class="myEntry cas13a"> | ||
+ | <li class="myTitel"> | ||
+ | <b> various Rnase alert experiments </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | with Noro Virus (10 ul total volume used for the experiment) </br> | ||
+ | |||
+ | </li> | ||
+ | <li> | ||
+ | with Q5 beta (10 ul total volume used for the experiment) | ||
+ | </li> | ||
+ | <li> | ||
+ | with Bacillus ( 10 ul total volume used for the experiment) and | ||
+ | </li> | ||
+ | <li> | ||
+ | with HCV and E.coli RNA (FINA method) samples (10 ul total volume used for the experiment) | ||
+ | </li> | ||
+ | <li> | ||
+ | Pipetting Scheme Norovirus and Q5 beta: </br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/a/a2/T--Munich--pic--platereader_pipScheme_03_10_norovirus_q5beta.png"> | ||
+ | </li> | ||
+ | <li> | ||
+ | Pipetting Scheme JVC and B. subtilis: </br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/e8/T--Munich--pic--platereader_pipScheme_03_10_hvc_bacillus.png"> | ||
+ | </li> | ||
+ | <li> | ||
+ | Pipetting Scheme E.coli RNA (FINA method): </br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/4/4e/T--Munich--pic--platereader_pipScheme_03_10_ecoli.png"> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | Bacillus, Noro virus and HCV seeem to work. However Q5 beta needs to be repeated and also HCV could be repeated! | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b> FINA extraction of RNA from E. coli with and without filters </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">FINA RNA extraction</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Jorge | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | Works, Target-sequence with PCR detected. | ||
+ | </li> | ||
+ | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
Line 6,224: | Line 6,591: | ||
<div class="date">Wednesday 4th</div> | <div class="date">Wednesday 4th</div> | ||
− | <ul class="myEntry other"> <!-- | + | |
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b> Bacillus RNA extraction </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Erika, Dawa | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | |||
+ | Resuspension Buffer (20 ml): | ||
+ | <li> | ||
+ | Sucrose (0.3 M) -> 2.1 g | ||
+ | </li> | ||
+ | <li> | ||
+ | Sodium Acetate (10 mM) -> 16.4 mg | ||
+ | |||
+ | </li> | ||
+ | <li> | ||
+ | Adjust pH to 4.2 | ||
+ | |||
+ | </li> | ||
+ | <li> | ||
+ | Stored at 4°C | ||
+ | |||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,241: | Line 6,642: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Send Lwa short colony 2-3 for sequencing with pseqLwa primer <!-- | + | Send Lwa short colony 2-3 for sequencing with pseqLwa primer <!--All--> |
</li> | </li> | ||
Line 6,248: | Line 6,649: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
Full read with no mutations. | Full read with no mutations. | ||
</li> | </li> | ||
</ul> | </ul> | ||
<div class="date">Thursday 5th</div> | <div class="date">Thursday 5th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry cas13a"> |
+ | <li class="myTitel"> | ||
+ | <b> RNaseAlert </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa, Rob, Erika | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | RNaseAlert reaction mix ( in glass fiber filter paper) lyophilisation. Introduction and first trial | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
+ | |||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,270: | Line 6,691: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Repeated Gibbson-Assembly for Lbu with 4:1 ratio<!-- | + | Repeated Gibbson-Assembly for Lbu with 4:1 ratio<!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 6,279: | Line 6,700: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
Empty plates => need to repeat PCR. | Empty plates => need to repeat PCR. | ||
Line 6,303: | Line 6,724: | ||
</ul> | </ul> | ||
<div class="date">Friday 6th</div> | <div class="date">Friday 6th</div> | ||
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b> RNA extraction from Bacillus overnight culture </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">Phenol-chlorophorm extraction</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Erika | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | following SIGMA Genosys Protocol from Step 1 to 7. Then, followed Lab´s protocol steps. | ||
+ | </li> | ||
+ | <li> | ||
+ | RNA sample stored at -80°C and needs to be quantified. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
Line 6,496: | Line 6,939: | ||
</ul> | </ul> | ||
<div class="date">Monday 9th</div> | <div class="date">Monday 9th</div> | ||
− | <ul class="myEntry cas13a"> <!-- | + | <ul class="myEntry other"> |
+ | <li class="myTitel"> | ||
+ | <b> Quantification of viral RNAs and Bacillus RNA extracted from last week </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNA urea gel</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry cas13a"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Lbu-Recloning with Gibbson-Assembly</b> <!-- | + | <b>Lbu-Recloning with Gibbson-Assembly</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR,Gibbson and Trafo in chem and e comp</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR,Gibbson and Trafo in chem and e comp</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,513: | Line 6,967: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | PCR for Gibbson with Biobrick fwd and rev primer. <!-- | + | PCR for Gibbson with Biobrick fwd and rev primer. <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 6,525: | Line 6,979: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
No colonies | No colonies | ||
</li> | </li> | ||
Line 6,556: | Line 7,010: | ||
</ul> | </ul> | ||
<div class="date">Tuesday 10th</div> | <div class="date">Tuesday 10th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR and Gibbson Assembly</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR and Gibbson Assembly</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,573: | Line 7,027: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | PCR of Lbu and psB1C3 as backbone <!-- | + | PCR of Lbu and psB1C3 as backbone <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 6,583: | Line 7,037: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
</li> | </li> | ||
Line 6,620: | Line 7,074: | ||
<div class="date">Wednesday 11th</div> | <div class="date">Wednesday 11th</div> | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry cas13a"> |
+ | <li class="myTitel"> | ||
+ | <b> Plate reader experiments </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa, Jorge | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | with Noro virus samples ( worked!) | ||
+ | </li> | ||
+ | <li> | ||
+ | with Bacillus ( seems to have Rnase contamination) | ||
+ | </li> | ||
+ | <li> | ||
+ | with the paper strips (glass fiber filter paper) 1.8 ul reaction mix pipetted with different amounts of invitro transcribed RNAs. | ||
+ | </li> | ||
+ | <li> | ||
+ | with E.coli RNA samples( heat lysed)- ( seems to have RNase contamination) | ||
+ | </li> | ||
+ | |||
+ | <li> | ||
+ | Pipetting Scheme 1: </br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/e/ea/T--Munich--pic--platereader_pipScheme_10_10_div1.png"> | ||
+ | </li> | ||
+ | <li> | ||
+ | Pipetting Scheme 2: </br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/c/c4/T--Munich--pic--platereader_pipScheme_10_10_div2.png"> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | |||
+ | </ul> | ||
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b>Coupling <i>In-Vitro Transcription</i> to RPA in Bulk and on Paper</b> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Coupling RPA to In-Vitro Transcription</a> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Sven | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Transcription efficiency when coupled to RPA was determined using a reaction without T7 RNA Polymerase as negative control. | ||
+ | The construct as template used was RNA Amp G-Block 200 bp double-stranded DNA using VF and VR as primers. | ||
+ | |||
+ | </li> | ||
+ | <li> | ||
+ | RPA-Tx and RPA-Tx on Paper according to protocol. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
+ | |||
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b>G-Block cloning of RNA-Amplification test plasmid via Restriction Cloning and Gibson Assembly</b> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">G-Block Cloning</a> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Sven | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Preparation: <br> | ||
+ | Solubilisation of G-Block to reach 10 ng/ul concentration. <br> | ||
+ | PCR reaction using VF and VR as primers resulted in 50 ul of a 24 ng/ul solution. <br> | ||
+ | </li> | ||
+ | <li> | ||
+ | Gibson Assembly: <br> | ||
+ | PCR reaction using pSB1A3_fw and pSB1A3_rev for backbone amplification of pSB1C3-GFP yielded 54 ng/ul. | ||
+ | Ratio for Gibson Assembly: 0.375 pmol Insert : 0.125 pmol backbone. Incubation for 15 minutes at 50 °C. | ||
+ | </li> | ||
+ | <li> | ||
+ | Restriction Cloning:<br> | ||
+ | Restriction of backbone and insert using EcoRI-HF and SpeI-HF in NEB Cutsmart Buffer and heat inactivation. <br> | ||
+ | Subsequent dephosphorylation of backbone using Antartic Phosphatase. <br> | ||
+ | T4 DNA Ligation with a ratio of 0.02 pmol backbone : 0.06 pmol insert. | ||
+ | </li> | ||
+ | <li> | ||
+ | Transformation of Ligation and Gibson Assembly into chemocompetent and electrocompetent Turbo cells. Plate-out on | ||
+ | chloramphenicol agar plates. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | The investigation of the agar plates the next day using UV-light revealed that the only plasmid that was taken up by the Turbo cells | ||
+ | was the initial pSB1C3-GFP plasmid. No positive inserts could be identified. | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning </b> <!-- | + | <b>Biobrick Recloning </b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of chem comp cells</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of chem comp cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,637: | Line 7,201: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Transformation of Turbo cells with Gibbson of 10.10.<!-- | + | Transformation of Turbo cells with Gibbson of 10.10.<!--All--> |
</li> | </li> | ||
Line 6,731: | Line 7,295: | ||
<div class="date">Thursday 12th</div> | <div class="date">Thursday 12th</div> | ||
− | <ul class="myEntry | + | <ul class="myEntry cas13a"> |
+ | <li class="myTitel"> | ||
+ | <b> Plate reader experiment with paper strips blocked ovenight with BSA </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | 1.8 ul of reaction mix pipetted in each paper with different amounts of in vitro target RNA. | ||
+ | </li> | ||
+ | <li> | ||
+ | The overnight blocked paper strips were rinsed with nuclease free water and then dried in the oven at 70 degree for 30 mins and then used for the experiments. | ||
+ | </li> | ||
+ | <li> | ||
+ | Pipetting scheme: <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/3/30/T--Munich--pic--platereader_pipScheme_12_10_paper.png"> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | Seems to work! Needs to be repeated for the confirmation of results. | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry other"> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b> | + | <b>UREA-PAGE of Coupling <i>In-Vitro Transcription</i> to RPA in Bulk and on Paper</b> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols"> | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Coupling RPA to In-Vitro Transcription</a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: | + | Participants: Sven |
</li> | </li> | ||
Line 6,748: | Line 7,342: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Transformation of BL21 with psB4A5 C-Term and psB1C3 N-Term <!-- | + | Transcription efficiency when coupled to RPA was determined using a reaction without T7 RNA Polymerase as negative control. |
+ | The construct as template used was RNA Amp G-Block 200 bp double-stranded DNA using VF and VR as primers. | ||
+ | |||
+ | </li> | ||
+ | <li> | ||
+ | RPA-Tx and RPA-Tx on Paper according to protocol. | ||
+ | </li> | ||
+ | <li> | ||
+ | UREA-PAGE Gel of Samples extracted via Phenol-Choloform-Extraction; both according to protocol. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | <ul> | ||
+ | <li>T7 RNA-Polymerase was, by accident, also added to the negative control of the RPA-Tx reaction on Paper. </li> | ||
+ | <li>First, one can see that the gel was overloaded. Also, it seems like there is additional bands at the expected size of around 150 bp.</li> | ||
+ | <li>Questionable is that this seems also to be the case for the negative Control of the RPA-Tx reaction in Bulk. </li> | ||
+ | <li>The experiment on paper, however, looks quite promising since it seems like there are distinct bands at the expected approximate size of the RNA transcript. Cas13a cleavage assay will provide evidence if these bands actually consist of the desired target RNA. | ||
+ | </li> | ||
+ | </ul> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/a/ad/T--Munich--Results_20171012_Urea_PAGE_RNA_RPA.png"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/c/c3/T--Munich--Results_20171012_Urea_PAGE_RNA_RPATx_bulk.png"> | ||
+ | </li> | ||
+ | </ul> | ||
+ | |||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
+ | <li class="myTitel"> | ||
+ | <b>Intein-Extein-Readout</b> <!----> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Max <!-- --> | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Transformation of BL21 with psB4A5 C-Term and psB1C3 N-Term <!--All--> | ||
</li> | </li> | ||
Line 6,755: | Line 7,392: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
No colonies for psB4A5 C-Term. | No colonies for psB4A5 C-Term. | ||
</li> | </li> | ||
Line 6,761: | Line 7,398: | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Col-PCR for Biobrick Recloning</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Col-PCR for Biobrick Recloning</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,778: | Line 7,415: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Col PCR of 6x N-Term in psB1C3, 16x C-Term in psB1C3, 14x Lbu in psB1C3 <!-- | + | Col PCR of 6x N-Term in psB1C3, 16x C-Term in psB1C3, 14x Lbu in psB1C3 <!--All--> |
</li> | </li> | ||
</ul> | </ul> | ||
Line 6,784: | Line 7,421: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
All negative. | All negative. | ||
</li> | </li> | ||
Line 6,834: | Line 7,471: | ||
</ul> | </ul> | ||
<div class="date">Friday 13th</div> | <div class="date">Friday 13th</div> | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry cas13a"> |
+ | <li class="myTitel"> | ||
+ | <b> Plate reader experiment with paper strips blocked ovenight with BSA </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Rob | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | |||
+ | |||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | 1.8 ul of reaction mix pipetted in each paper with different amounts of in vitro target RNA. | ||
+ | </li> | ||
+ | <li> | ||
+ | The overnight blocked paper strips were rinsed with nuclease free water and then dried in the oven at 70 degree for 30 mins and then used for the experiments. (Results seem to work!) | ||
+ | </li> | ||
+ | </ul> | ||
+ | <li> | ||
+ | Pipetting scheme: <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/6/6b/T--Munich--pic--platereader_pipScheme_14_10_paper.png"> | ||
+ | </li> | ||
+ | </li> | ||
+ | |||
+ | </ul> | ||
+ | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR with Q5-Polymerase</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">PCR with Q5-Polymerase</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,851: | Line 7,517: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Repeated PCR of N and C-term part for Recloning <!-- | + | Repeated PCR of N and C-term part for Recloning <!--All--> |
</li> | </li> | ||
</ul> | </ul> | ||
Line 6,885: | Line 7,551: | ||
</ul> | </ul> | ||
<div class="date">Saturday 14th</div> | <div class="date">Saturday 14th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest Gibbson-Assembly and Transformation</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Restriction digest Gibbson-Assembly and Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,902: | Line 7,568: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | DpnI digest of PCR from 31.10.17 <!-- | + | DpnI digest of PCR from 31.10.17 <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 6,917: | Line 7,583: | ||
</ul> | </ul> | ||
<div class="date">Sunday 15th</div> | <div class="date">Sunday 15th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry cas13a"> |
+ | <li class="myTitel"> | ||
+ | <b> RNA extraction of E. coli and Bacillus + plate reader experiment</b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa, Jorge, Erika | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Notes: | ||
+ | <ul> | ||
+ | <li> | ||
+ | HCV assay and Q5beta in Fluorostar and Clariostar for 1 hour each (HCV assay worked!) | ||
+ | </li> | ||
+ | <li> | ||
+ | RNA urea gel for RNA extracted from E.coli and Bacillus | ||
+ | </li> | ||
+ | <li> | ||
+ | Plate reader experiment of RNA extracted from the E.coli in Fluorostar for 2 hours | ||
+ | </li> | ||
+ | <li> | ||
+ | Pipetting scheme: <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/f/f1/T--Munich--pic--platereader_pipScheme_15_10_hvc.png" | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,934: | Line 7,629: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Repeated Transformation of Bl21 star with psB4A5-Split-Intein-C-Term <!-- | + | Repeated Transformation of Bl21 star with psB4A5-Split-Intein-C-Term <!--All--> |
</li> | </li> | ||
</ul> | </ul> | ||
Line 6,940: | Line 7,635: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
No colonies.=> Transformation in other expression strains | No colonies.=> Transformation in other expression strains | ||
</li> | </li> | ||
Line 6,946: | Line 7,641: | ||
<div class="date">Monday 16th</div> | <div class="date">Monday 16th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry cas13a"> |
+ | <li class="myTitel"> | ||
+ | <b> Plate reader experiment with different concentrations of RNA in vitro targets, positive and negative controls. Plus assay with cas13a with TDPs and assay with RPA paper samples. | ||
+ | </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Pipetting scheme: <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/b/bc/T--Munich--pic--platereader_pipScheme_16_10_paper.png" > | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Transformation of e. comp cells</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 6,963: | Line 7,674: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Transformed all available Bl21 strains with psB4A5-C-Term <!-- | + | Transformed all available Bl21 strains with psB4A5-C-Term <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 6,975: | Line 7,686: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
No colonies for any BL21 strain. The Turbo cells grew fine. => Expression seems to be toxic. | No colonies for any BL21 strain. The Turbo cells grew fine. => Expression seems to be toxic. | ||
</li> | </li> | ||
Line 7,012: | Line 7,723: | ||
</ul> | </ul> | ||
<div class="date">Tuesday 17th</div> | <div class="date">Tuesday 17th</div> | ||
− | <ul class="myEntry readout"> <!-- | + | <ul class="myEntry cas13a"> |
+ | <li class="myTitel"> | ||
+ | <b> Plate reader experiment with different concentrations of RNA in vitro targets, positive and negative controls. Plus assay with cas13a with TDPs and assay with RPA paper samples. | ||
+ | </b> | ||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="#">RNase activity</a> | ||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Dawa | ||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | <ul> | ||
+ | <li> | ||
+ | Pipetting Scheme B. subtilis:<br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/9/91/T--Munich--pic--platereader_pipScheme_17_10_bacillus.png"> | ||
+ | </li> | ||
+ | <li> | ||
+ | Pipetting Scheme RPA paper:<br> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/1/1b/T--Munich--pic--platereader_pipScheme_17_10_paper.png"> | ||
+ | </li> | ||
+ | |||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul class="myEntry readout"> <!--, existing categories are Cas13a,readout,other--> | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Intein-Extein-Readout</b> <!-- | + | <b>Intein-Extein-Readout</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">-</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 7,029: | Line 7,765: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Midipreparation of psB4A5-C-Term with Macherey-Nagel Xtra Midi <!-- | + | Midipreparation of psB4A5-C-Term with Macherey-Nagel Xtra Midi <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 7,041: | Line 7,777: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
In vitro expression was not done, due to lack of time. | In vitro expression was not done, due to lack of time. | ||
</li> | </li> | ||
</ul> | </ul> | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Q5-PCR, Gibbson-Assembly and Transformation</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Q5-PCR, Gibbson-Assembly and Transformation</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 7,063: | Line 7,799: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | PCR of all GFP degradation tag library variants with Biobrick sequence binding primers, for transfer from psB1A3 to psB1C3. <!-- | + | PCR of all GFP degradation tag library variants with Biobrick sequence binding primers, for transfer from psB1A3 to psB1C3. <!--All--> |
</li> | </li> | ||
<li> | <li> | ||
Line 7,073: | Line 7,809: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
</li> | </li> | ||
Line 7,107: | Line 7,843: | ||
</ul> | </ul> | ||
<div class="date">Wednesday 18th</div> | <div class="date">Wednesday 18th</div> | ||
− | <ul class="myEntry other"> <!-- | + | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b>Biobrick Recloning</b> <!-- | + | <b>Biobrick Recloning</b> <!----> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Overnight culture</a> <!-- | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Overnight culture</a> <!--(NOT the href=..., the "Total RNA Purific...")--> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: Max <!-- | + | Participants: Max <!-- --> |
</li> | </li> | ||
Line 7,124: | Line 7,860: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Picked 2 green colonies from each plate for overnight culture <!-- | + | Picked 2 green colonies from each plate for overnight culture <!--All--> |
</li> | </li> | ||
Line 7,160: | Line 7,896: | ||
</ul> | </ul> | ||
<div class="date">Thursday 19th</div> | <div class="date">Thursday 19th</div> | ||
− | + | <ul class="myEntry other"> | |
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b> | + | <b>G-Block cloning of RNA-Amplification test plasmid via Restriction Cloning and Gibson Assembly</b> |
</li> | </li> | ||
<li class="myProtocol"> | <li class="myProtocol"> | ||
− | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols"> | + | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">G-Block Cloning</a> |
</li> | </li> | ||
<li class="myParticipants"> | <li class="myParticipants"> | ||
− | Participants: | + | Participants: Sven |
</li> | </li> | ||
Line 7,177: | Line 7,913: | ||
<ul> | <ul> | ||
<li> | <li> | ||
− | Send samples from degradation tag library for sequencing <!-- | + | Preparation: <br> |
+ | PCR reaction of G-Block using VF and VR as primers resulted in 50 ul of a 27 ng/ul solution. <br> | ||
+ | </li> | ||
+ | <li> | ||
+ | Gibson Assembly: <br> | ||
+ | PCR reaction using pSB1A3_fw and pSB1A3_rev for backbone amplification of pSB1C3-GFP yielded 52 ng/ul. | ||
+ | Ratio for Gibson Assembly: 0.375 pmol Insert : 0.125 pmol backbone. Incubation for 15 minutes at 50 °C. | ||
+ | </li> | ||
+ | <li> | ||
+ | Restriction Cloning:<br> | ||
+ | Restriction of backbone and insert using EcoRI-HF and SpeI-HF in NEB Cutsmart Buffer and heat inactivation. <br> | ||
+ | Simultanous dephosphorylation of backbone using Antartic Phosphatase. <br> | ||
+ | T4 DNA Ligation with a ratio of 0.02 pmol backbone : 0.06 pmol insert. | ||
+ | </li> | ||
+ | <li> | ||
+ | Transformation of Ligation and Gibson Assembly into chemocompetent and electrocompetent Turbo cells. Plate-out on | ||
+ | chloramphenicol agar plates. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | <ul> | ||
+ | <li>The investigation of the agar plates the next day using UV-light showed that for the Gibson Assembly, positive clones were visible. </li> | ||
+ | <li>Colony PCR was performed and showed inserts for several plasmids.</li> | ||
+ | <li>Overnight cultures were inocculated for sequencing.</li> | ||
+ | </ul> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/6/68/T--Munich--Results_20171019_Colony_PCR_RNA_amp.png"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/0/01/T--Munich--Results_20171019_RNA_M_AMP.png"> | ||
+ | </li> | ||
+ | |||
+ | </ul> | ||
+ | <ul class="myEntry other"> <!--, existing categories are Cas13a,readout,other--> | ||
+ | <li class="myTitel"> | ||
+ | <b>Biobrick Recloning</b> <!----> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">Sequencing</a> <!--(NOT the href=..., the "Total RNA Purific...")--> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Max <!-- --> | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Send samples from degradation tag library for sequencing <!--All--> | ||
</li> | </li> | ||
</ul> | </ul> | ||
Line 7,183: | Line 7,968: | ||
<li class="myResults"> | <li class="myResults"> | ||
<h4>Results</h4> | <h4>Results</h4> | ||
− | <!-- | + | <!----> |
pdt2-E 2 was positive | pdt2-E 2 was positive | ||
</li> | </li> | ||
Line 7,189: | Line 7,974: | ||
<div class="date">Friday 20th</div> | <div class="date">Friday 20th</div> | ||
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b>G-Block cloning of RNA-Amplification test plasmid</b> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">G-Block Cloning</a> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Sven | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Mini-prep of previously inocculated overnight cultures and sent to sequencing. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | <ul> | ||
+ | <li>The investigation of the agar plates the next day using UV-light showed that for the Gibson Assembly, positive clones were visible. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
Line 7,268: | Line 8,082: | ||
<li class="myTitel"> | <li class="myTitel"> | ||
− | <b> | + | <b>In vitro transcription of crRNA Lwa at 37 C overnight</b> |
</li> | </li> | ||
Line 7,287: | Line 8,101: | ||
</ul> | </ul> | ||
<div class="date">Sunday 22nd</div> | <div class="date">Sunday 22nd</div> | ||
+ | <ul class="myEntry other"> | ||
+ | <li class="myTitel"> | ||
+ | <b>RPA on PDMS Chip</b> | ||
+ | |||
+ | </li> | ||
+ | <li class="myProtocol"> | ||
+ | Protocol: <a href="https://2017.igem.org/Team:Munich/Protocols">RPA on Paper</a> | ||
+ | |||
+ | </li> | ||
+ | <li class="myParticipants"> | ||
+ | Participants: Sven | ||
+ | |||
+ | </li> | ||
+ | <li class="myObservations"> | ||
+ | Observations: | ||
+ | <ul> | ||
+ | <li> | ||
+ | Since the PDMS chip was leaky, no real sample could be extracted from it. | ||
+ | </li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li class="myResults"> | ||
+ | <h4>Results</h4> | ||
+ | <ul> | ||
+ | <li> Positive Controls as well as colony-PCR using RPA showed clear bands on a 2%-agarose gel. The RPA performed on Chip, however, was not showing any bands though Nanodrop concentration showed 27 ng/ul in the sample. </li> | ||
+ | </ul> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/2f/T--Munich--Results_20171022_RPA_on_Chip.png"> | ||
+ | </li> | ||
+ | </ul> | ||
<ul class="myEntry readout"> | <ul class="myEntry readout"> | ||
Line 7,835: | Line 8,678: | ||
</li> | </li> | ||
</ul> | </ul> | ||
− | + | ||
<div class="date">Tuesday 31st</div> | <div class="date">Tuesday 31st</div> | ||
Line 7,843: | Line 8,686: | ||
<div class="three wide column" id="monthsList"> | <div class="three wide column" id="monthsList"> | ||
<ul> | <ul> | ||
− | |||
<li><a href="#April">April</a></li> | <li><a href="#April">April</a></li> | ||
<li><a href="#May">May</a></li> | <li><a href="#May">May</a></li> |
Latest revision as of 11:45, 1 November 2017
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|