Difference between revisions of "Team:Potsdam/notebook/lowcopy"

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   </tr>
 
   </tr>
 
</table>
 
</table>
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<br> <br>
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<b> Wednesday, 08/16/17 </b>
 +
<br> <br>
 +
1. Gel run of psB4A5 gradient PCR (A,B,C,0.1,1,10)<br>
 +
<div style="text-align: justify; margin-left:20px">
 +
- run at 120 V a 40 min -> only 2 bands at well 1A <br>
 +
- run at 120 V another 30 min -> lost DNA from gel <br>
 +
- <b>1A (60°C/1:100 DNA) might have worked so I did a PCR purification for this one </b></div>
 +
 +
2. Gel purification of dCAs9 A + B fragments and 1A-psB4A5 linearized plasmid <br>
 +
<div style="text-align: justify; margin-left:20px">
 +
- used Promega PCR/Gel purification kit: final concentration pSB4A5 18 ng/uL <br>
 +
 +
https://www.promega.de/resources/protocols/technical-bulletins/101/wizard-sv-gel-and-pcr-cleanup-system-protocol/ <br>
 +
 +
<b>IMPORTANT: We use AE-buffer for elution in the last step (50 uL) instead of water!</b></div>
 +
 +
3. Fusion PCR of dCAs9 fragments A + B <br>
 +
<div style="text-align: justify; margin-left:20px">
 +
- Primer used: Q5 + Q6 </div><br><br>
 +
<table>
 +
  <tr>
 +
    <th  width="50%" align="center"><b>component</b></th>
 +
    <th  width="50%" align="center"><b>volume in µl</b></th>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">Q5-MM</td>
 +
    <td align="center">12,5</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">DNA</td>
 +
    <td align="center">2(1 pro fragment)</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">H<sub>2</sub>O</td>
 +
    <td align="center">10,5</td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">final volume</td>
 +
    <td align="center">25</td>
 +
  </tr>
 +
</table>
 +
<br> <br>
 +
 +
<b>IMPORTANT: Fusion PCR done like team munich!</b><br>
 +
 +
https://static.igem.org/mediawiki/2014/c/c9/LMU_Munich14_FusionPCR.pdf<br>
 +
<div style="text-align: justify; margin-left:20px">
 +
- first made a PCR mix of 25 uL without primers<br>
 +
- then run mix in thermocycler with programm (fusion pcr)<br>
 +
- in the middle there is a hold step in the programm<br>
 +
- you then add the primers mixed with buffer (5 uL total volume) for an endvolume of 30 uL<br>
 +
- IG_C_Q19/20 and Q24 resuspended auf 100 uM and diluted to 10 uM </div>
 
<br> <br>
 
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</p>
 
</p>

Revision as of 11:18, 1 November 2017

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Lab book

We splitted our labbook into the following parts:



Friday, 07/21/17

- took pSB4A5 backbone out of DNA distribution Kit and transformed into E.coli
(rest from Kit was put in -20 °C)

Tuesday, 07/25/17

- Inoculated LB media with psB4A5

Wednesday, 07/26/17

- Miniprep with Promega “Plus SV Miniprep DNA Purification System”
- 2 approaches with psB4A5
- final concentration:
- approach 1: 30 ng/ul
- approach 2: 40 ng/ul


Thursday, 08/10/17

- glycerol stock (30%) of IG_C_P1_G (pSB4A5)
- prepared 10 mL preculture with pdCas9-Bacteria E.coli from plate from Fabian for Miniprep


Monday, 08/14/17

- primer resuspended to 100 uM (except Q8):

primer volume in µl
Q5 354
Q6 957
Q7 749
Q8 657 (83,1 uM)
Q15 956
Q16 1455
Q17 1693
Q18 1440
- diluted all primers to 10 uM in separate aliquotes
- prepareed 3 separate PCRs with primer pairs:
Q5/Q8 (Eppi A), Q6/Q7 (Eppi B), Q15/Q16 (Eppi c)
- pipetting scheme:
Q5 2X MasterMix: 5 uL
10 uM Primer: each 0,5 uL
Template: 1 uL of 1:100 diluted Miniprep (about 0,5 ng)
ddH2O: 3 uL
total: 10 uL
- cycling conditions (prog01 in Epepndorf Mastercycler nexus gradient (Torsten):

temperature time in sec
98 °C 30
98 °C 10
64 °C 20
72 °C 110
72 °C 120
- after PCR: gel electrophoresis as follows:
- 2.5 uL DNA + 2uL Loading Dye + 7.5 uL (total 12 uL)
- ran gel at 120 V for 40 minutes
gel picture:

- order of pockets: ABC, A and B are correct size, C did not work well, there is a band at the desired result and one at the unopened plasmid (and several other stuff) so PCR has to be done again
- dCas9 fragments look good and can be worked with tomorrow


Tuesday, 08/15/17

1. DpnI digestion of dCas9 fragments A + B

component volume in µl
DpnI 2,6
DNA 6,5
cutsmart-Buffer 5,0
H2O 35,9
final volume 50
- Duration: 1 hour

IMPORTANT: Fabian told me you can just add about 1 uL DpnI to the reaction-mix

2. Gel run of dCas9 A + B
- let it run on gel for purification
- 140 V a 40 min
- cut out was done by Natalie and Pauline

3. Linearization PCR of psB4A5 (second atempt) via gradient PCR
- made new PCR using Q15/Q16 primers and psB4A5 plasmid fpr linearization
- used 3 different DNA concentrations and 3 different annealing temperatures (9 reactions total)

component 1 reaction:
volume in µl		 10 reaction MM:
volume in µl
HiQ 5 50
primer 1,25 12,5
DNA variable (1 µl) variable
H2O 17,5 175
final volume 25 250
- annealing temperatures: A = 60°C/B = 64,5°C/C = 68°C
- DNA concentrations: 0,1 = 1:1000 / 1 = 1:100 / 10 = 1:10
Scheme:
0,1 A 0,1 B 0,1 C
1 A 1 B 1 C
10 A 10 B 10 C


Wednesday, 08/16/17

1. Gel run of psB4A5 gradient PCR (A,B,C,0.1,1,10)
- run at 120 V a 40 min -> only 2 bands at well 1A
- run at 120 V another 30 min -> lost DNA from gel
- 1A (60°C/1:100 DNA) might have worked so I did a PCR purification for this one
2. Gel purification of dCAs9 A + B fragments and 1A-psB4A5 linearized plasmid
- used Promega PCR/Gel purification kit: final concentration pSB4A5 18 ng/uL
https://www.promega.de/resources/protocols/technical-bulletins/101/wizard-sv-gel-and-pcr-cleanup-system-protocol/
IMPORTANT: We use AE-buffer for elution in the last step (50 uL) instead of water!
3. Fusion PCR of dCAs9 fragments A + B
- Primer used: Q5 + Q6


component volume in µl
Q5-MM 12,5
DNA 2(1 pro fragment)
H2O 10,5
final volume 25


IMPORTANT: Fusion PCR done like team munich!
https://static.igem.org/mediawiki/2014/c/c9/LMU_Munich14_FusionPCR.pdf
- first made a PCR mix of 25 uL without primers
- then run mix in thermocycler with programm (fusion pcr)
- in the middle there is a hold step in the programm
- you then add the primers mixed with buffer (5 uL total volume) for an endvolume of 30 uL
- IG_C_Q19/20 and Q24 resuspended auf 100 uM and diluted to 10 uM