Difference between revisions of "Team:Uppsala/Experiments"

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Another heat kill around at 80 °C for 20 minutes was then performed.<br>
 
Another heat kill around at 80 °C for 20 minutes was then performed.<br>
 
<br>
 
<br>
−
For the transformation step see the Phusion PCR, Gibson Assembly and Transformation protocol (link to the mentioned protocol).
+
For the transformation step see the <a href="#phusion">Phusion PCR, Gibson Assembly and Transformation</a> protocol.
 
</p>
 
</p>
  
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</div>
 
</div>
  
−
<div class="container-fluid txet content" style="padding:0%;">
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<div class="container-fluid txet content" style="padding:0%;" id="phusion">
−
<div class="potroloc"><h1>Lorem ipsum</h1></div>
+
<div class="potroloc"><h1>Phusion PCR, Gibson assembly and Transformation</h1></div>
 
<img src="https://static.igem.org/mediawiki/2017/f/f3/Uppsala_protocol_top.svg" class="img-responsive" style="width:100%;"> </img>
 
<img src="https://static.igem.org/mediawiki/2017/f/f3/Uppsala_protocol_top.svg" class="img-responsive" style="width:100%;"> </img>
 
 
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<div class="row ">
 
<div class="row ">
 
<div class="col-sm-6">
 
<div class="col-sm-6">
 +
<h3>1. Phusion PCR</h3>
 +
 
<p>
 
<p>
−
Lorem ipsum dolor sit amet, consectetur adipiscing elit.
+
Total reaction volume 50 µL<br>
−
Ut lobortis, sem a sagittis rhoncus, enim metus sollicitudin purus,
+
Amount of plasmid per reaction ~30 pg
−
quis porttitor enim leo at nunc. Aenean maximus metus in feugiat viverra.
+
</p>
−
  Aenean a luctus lectus. Aenean sollicitudin ex sed ipsum accumsan aliquam.
+
−
  Pellentesque ultricies aliquam ipsum, sed fringilla nunc hendrerit vel.
+
−
Proin sagittis feugiat nunc at pulvinar. Fusce id pellentesque tellus,
+
−
tempor tincidunt ipsum. Sed dignissim mattis dolor a placerat.
+
−
Morbi leo libero, lobortis volutpat rutrum ac, volutpat ac purus.
+
−
Aenean finibus libero in metus fermentum commodo. Curabitur feugiat
+
−
enim nec cursus hendrerit. Morbi vel nibh elementum, eleifend magna
+
−
cursus, facilisis massa. Nulla lobortis tincidunt venenatis. Vestibulum
+
−
id nibh est. Mauris lobortis felis quis libero facilisis, sit
+
−
amet convallis ipsum pretium. Suspendisse potenti.</p>
+
  
−
<p>Aenean vulputate tellus id egestas volutpat.
+
<p><b>Phusion PCR reaction mixture:</b></p>
−
Vivamus consectetur fermentum varius. Suspendisse sed diam et erat
+
<table>
−
ultricies tincidunt. Etiam tempus, libero non varius tempor, lacus
+
  <tr>
−
tortor cursus metus, ac semper metus dui eu tellus. Mauris maximus
+
    <td>Plasmid</td>
−
odio urna, vitae eleifend turpis bibendum a. Quisque nec posuere urna.
+
    <td>…. µL (~30 pg)</td>
−
Aliquam enim neque, convallis ut dignissim sit amet, mattis sit amet
+
  </tr>
−
urna. Etiam ultricies risus vitae elit gravida, ac aliquam nibh
+
  <tr>
−
venenatis. Vivamus posuere eget tellus ullamcorper condimentum.</p>
+
    <td>5X Phusion HF Buffer</td>
 +
    <td>10 µL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>dNTP (10 mM) </td>
 +
    <td><1 µL/td>
 +
  </tr>
 +
  <tr>
 +
    <td>Reverse Primer (10 µM)</td>
 +
    <td>2.5 µL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Forward Primer (10 µM)</td>
 +
    <td>2.5 µL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Phusion DNA pol </td>
 +
    <td>0.5 µL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>DMSO</td>
 +
    <td>1.5 µL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>H<sub>2</sub>O</td>
 +
    <td>up to 50 µL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Total volume</td>
 +
    <td>50 µL</td>
 +
  </tr>
 +
</table>
  
−
<p>Duis rhoncus, ex ac varius aliquet, justo justo commodo augue,
+
<p><b>PCR reaction conditions:</b></p>
−
vel dictum enim tortor id elit. Duis sagittis, enim non sodales viverra,
+
<table>
−
lacus purus congue quam, nec tempus metus mi vitae sem. Vivamus dapibus
+
  <tr>
−
id leo vitae volutpat. Sed vulputate fermentum facilisis.
+
    <td>1)  Initial denaturation: </td>
−
Nullam gravida dictum accumsan. Etiam tincidunt vehicula tincidunt.
+
    <td>98 °C for 30 seconds </td>
−
Mauris faucibus ultrices purus, at cursus erat. Sed at varius dui.
+
  </tr>
−
Nunc accumsan nunc congue volutpat faucibus. Praesent mollis
+
  <tr>
−
tincidunt auctor. Ut aliquet risus eros, nec accumsan sem congue quis.
+
    <td>2)    Denaturation in following cycles: </td>
−
Vivamus pulvinar aliquet turpis, ac sodales arcu feugiat id.
+
    <td>98 °C for 10 seconds</td>
−
Sed ullamcorper mauris purus. Sed cursus quam iaculis metus
+
  </tr>
−
efficitur, eu auctor ipsum posuere.</p>
+
  <tr>
 +
    <td>3)  Primer annealing: </td>
 +
    <td>Temperature gradient between 58–65 °C for 30 seconds</td>
 +
  </tr>
 +
  <tr>
 +
    <td>4)  Extension:</td>
 +
    <td>72 °C 1.5 minutes</td>
 +
  </tr>
 +
  <tr>
 +
    <td>5)      Final DNA synthesis:</td>
 +
    <td>72 °C for 5 minutes</td>
 +
  </tr>
 +
</table>
  
−
<p>Curabitur porta sagittis ultricies.
+
<p>
−
Suspendisse dictum iaculis justo. Fusce consequat tristique mi,
+
Steps 2–4: 25 cycles<br>
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vel tempus neque bibendum ac. Ut ac dui risus. Ut mattis laoreet
+
Lid temperature at 105 °C
−
est, eu venenatis ligula vehicula ut. Donec ex tellus,
+
</p>
−
auctor quis arcu laoreet, rutrum facilisis odio. Donec condimentum,
+
−
felis id ultrices dictum, elit nunc fermentum neque,
+
−
vel tempor nulla ex sit amet neque. Aenean semper turpis nec erat sodales semper.</p>
+
  
−
<p>Mauris nulla libero, rhoncus eget ante at, sollicitudin posuere ante.
+
<h3>2. DpnI Treatment of the PCR Fragment</h3>
−
Class aptent taciti sociosqu ad litora torquent per conubia nostra,
+
 
−
per inceptos himenaeos. Vivamus felis risus, faucibus at diam id,
+
<p>
−
accumsan ultricies tortor. Aenean vitae libero massa.
+
To the 50 µL PCR reaction mix from step 1 add:<br>
−
  Donec pellentesque mi non libero varius, ac luctus magna lobortis.
+
  <br>
−
Nulla vehicula eros erat, ac pharetra enim fringilla vel.
+
2.4 µL “FastDigest” DpnI<br>
−
Nullam euismod pellentesque ante, a elementum mi efficitur non.
+
6 µL 10X “FastDigest” Buffer<br>
−
Maecenas lobortis malesuada leo, eget cursus dui sodales a.
+
<br>
−
Quisque ullamcorper porttitor lacus, ac dictum justo faucibus eget.
+
1) Incubate reaction mixture at 37 °C for 1 hour<br>
−
Vestibulum purus lorem, vehicula vel vehicula sit amet,
+
2) Purify the DNA in the mixture with “PureLink Quick PCR Purification Kit” (Invitrogen). Milliq was used instead of TE buffer when eluting the DNA from the spin column in the final step of the DNA purification. The optional washing step was skipped<br>
−
sagittis non leo. Proin consectetur sollicitudin est et laoreet.  
+
3) Measure the DNA concentration on Nanodrop in units of ng/µL. 2 µL was used for each measurement<br>
−
Suspendisse potenti. Cras nec pretium ligula. Praesent placerat
+
4) Store the DNA at -20 °C until further use
−
ipsum leo, mattis imperdiet nisi imperdiet feugiat.
+
 
−
</p>
+
</p>
 +
 
 +
 
  </div>
 
  </div>
 
<div class="col-sm-6">
 
<div class="col-sm-6">
 +
<h3>3. Gibson Assembly Reaction</h3>
 
<p>
 
<p>
−
Curabitur porta sagittis ultricies.
+
1. Mix PCR fragment from the plasmid and the Gibson fragment containing the insert. The total volume is 20 µL:<br>
−
Suspendisse dictum iaculis justo. Fusce consequat tristique mi,
+
<br>
−
vel tempus neque bibendum ac. Ut ac dui risus. Ut mattis laoreet
+
Mix i) the PCR fragment made from the plasmid and ii) the Gibson fragment containing the gene of interest. Total volume of the Gibson reaction mix is 20 uL :</p>
−
est, eu venenatis ligula vehicula ut. Donec ex tellus,
+
 
−
auctor quis arcu laoreet, rutrum facilisis odio. Donec condimentum,
+
<table>
−
felis id ultrices dictum, elit nunc fermentum neque,
+
  <tr>
−
vel tempor nulla ex sit amet neque. Aenean semper turpis nec erat sodales semper.
+
    <td>Plasmid PCR fragment </td>
−
</p>
+
    <td>.… µL (~0.02 pmol)</td>
−
<img src="https://www.mathsisfun.com/data/images/histogram.gif"></img>
+
  </tr>
 +
  <tr>
 +
    <td>Gibson fragment</td>
 +
    <td>.… µL (~0.06 pmol)</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Gibson Assembly Master Mix (2x)</td>
 +
    <td>10 µL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>H<sub>2</sub>O</td>
 +
    <td>up to 20 µL</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Total volume</td>
 +
    <td>20 µL</td>
 +
  </tr>
 +
</table>
 +
 
 +
<p>
 +
2. Incubate the mixture at 50 °C for 1 hour
 +
</p>
 +
 
 +
 
 +
<h3>4. Transformation</h3>
 +
 
 +
<p>
 +
1. Take out competent cells (we used  Escherichia Coli TOP10 for cloning and E. coli BL21 (DE3*) for protein expression from Invitrogen) from the -80 °C and thaw them on ice from approximately 15 minutes.<br>
 +
2. Add 2 µL of the Gibson mixture/Ligation mixture to the competent cells or ~7.5 ng of gBlocks.<br>
 +
3. Incubate on ice for 30 minutes.<br>
 +
4. Incubate at 42 °C for 30 seconds (water bath).<br>
 +
5. Incubate on ice for 5 minutes.<br>
 +
6. Add 200 µL of LB to the mixture and incubate it at 37 °C with 200 rpm for 1 hour.<br>
 +
7. Plate the mixtures on chloramphenicol plates (200 µL 1:1 dilution and 200 µL 1:100 diluted with LB). Beads were used to spread the mixture on the plates (10–20 beads).<br>
 +
8. Incubate plates overnight at 37 °C.
 +
 
 +
</p>
 +
 
 
</div>
 
</div>
 
</div>
 
</div>
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<div class="col-sm-6">
 
<div class="col-sm-6">
 
 
 +
 +
<p><b></b></p>
 +
<table>
 +
  <tr>
 +
    <td></td>
 +
    <td></td>
 +
  </tr>
 +
</table>
 
                 </div>
 
                 </div>
 
<div class="col-sm-6">
 
<div class="col-sm-6">

Revision as of 17:38, 30 October 2017

3A Assembly

1. Digestion

A 25 µL master mix was prepared for both of the inserts. The backbone plasmid was already precut, but to cut backbone plasmids EcoRI-HF and PstI would be used (the same volume).

Enzyme Master Mix for the first insert:

NEB Buffer 2 5 µL
EcoRI-HF 0.5 µL
SpeI 0.5 µL
Milliq 19 µL

4 µL of the master mix was then mixed with 4 µL of the enzyme.

Enzyme Master Mix for the second insert:

NEB Buffer 2 5 µL
XBaI 0.5 µL
PstI 0.5 µL
Milliq 19 µL

4 µL of the master mix was then mixed with 4 µL of the enzyme.

Both digestion mixtures were digested at 37 °C for 30 minutes. The mixtures were then left at 80 °C for 20 minutes to heat kill the enzymes. These steps were performed in a PCR machine.

2. Ligation

In this step it is important to have equimolar amount of cut plasmid backbone and digest product. In our case the length of everything was very similar, therefore the same volumes could be used, however that can always be done. The total reaction volume is 10 µL

Ligation Mixture:

Plasmid backbone 2 µL
Enzyme insert cut at E and S 2 µL
Enzyme insert cut at X and P 2 µL
T4 DNA Ligase Buffer 1 µL
T4 DNA Ligase 0.5 µL
MilliQ 2.5 µL

The mixture was then ligated at 16 °C for 30 minutes. Another heat kill around at 80 °C for 20 minutes was then performed.

For the transformation step see the Phusion PCR, Gibson Assembly and Transformation protocol.

Phusion PCR, Gibson assembly and Transformation

1. Phusion PCR

Total reaction volume 50 µL
Amount of plasmid per reaction ~30 pg

Phusion PCR reaction mixture:

Plasmid …. µL (~30 pg)
5X Phusion HF Buffer 10 µL
dNTP (10 mM) <1 µL/td>
Reverse Primer (10 µM) 2.5 µL
Forward Primer (10 µM) 2.5 µL
Phusion DNA pol 0.5 µL
DMSO 1.5 µL
H2O up to 50 µL
Total volume 50 µL

PCR reaction conditions:

1) Initial denaturation: 98 °C for 30 seconds
2) Denaturation in following cycles: 98 °C for 10 seconds
3) Primer annealing: Temperature gradient between 58–65 °C for 30 seconds
4) Extension: 72 °C 1.5 minutes
5) Final DNA synthesis: 72 °C for 5 minutes

Steps 2–4: 25 cycles
Lid temperature at 105 °C

2. DpnI Treatment of the PCR Fragment

To the 50 µL PCR reaction mix from step 1 add:

2.4 µL “FastDigest” DpnI
6 µL 10X “FastDigest” Buffer

1) Incubate reaction mixture at 37 °C for 1 hour
2) Purify the DNA in the mixture with “PureLink Quick PCR Purification Kit” (Invitrogen). Milliq was used instead of TE buffer when eluting the DNA from the spin column in the final step of the DNA purification. The optional washing step was skipped
3) Measure the DNA concentration on Nanodrop in units of ng/µL. 2 µL was used for each measurement
4) Store the DNA at -20 °C until further use

3. Gibson Assembly Reaction

1. Mix PCR fragment from the plasmid and the Gibson fragment containing the insert. The total volume is 20 µL:

Mix i) the PCR fragment made from the plasmid and ii) the Gibson fragment containing the gene of interest. Total volume of the Gibson reaction mix is 20 uL :

Plasmid PCR fragment .… µL (~0.02 pmol)
Gibson fragment .… µL (~0.06 pmol)
Gibson Assembly Master Mix (2x) 10 µL
H2O up to 20 µL
Total volume 20 µL

2. Incubate the mixture at 50 °C for 1 hour

4. Transformation

1. Take out competent cells (we used Escherichia Coli TOP10 for cloning and E. coli BL21 (DE3*) for protein expression from Invitrogen) from the -80 °C and thaw them on ice from approximately 15 minutes.
2. Add 2 µL of the Gibson mixture/Ligation mixture to the competent cells or ~7.5 ng of gBlocks.
3. Incubate on ice for 30 minutes.
4. Incubate at 42 °C for 30 seconds (water bath).
5. Incubate on ice for 5 minutes.
6. Add 200 µL of LB to the mixture and incubate it at 37 °C with 200 rpm for 1 hour.
7. Plate the mixtures on chloramphenicol plates (200 µL 1:1 dilution and 200 µL 1:100 diluted with LB). Beads were used to spread the mixture on the plates (10–20 beads).
8. Incubate plates overnight at 37 °C.