Difference between revisions of "Team:Warwick/Project5"

Week 1

We brainstormed ideas of what we can do with the project, more specifically and also split into smaller groups of modellers and biologists (although we had daily meetings). We spent a lot of time coming up with and optimising DNA sequences for zinc fingers.

Week 2

We marked plates, making 8 of streptomycin and chloramphenicol and 3 of chloramphenicol. We made the cells electrocompetent following lab protocol. The cells grew!
We were taught about cloning/ Gibson assembly and PCR. We did 4 mini-preps, electropheresis and several nanodrops.

Week 3

We grew up MG1655 (Z1) cells.
Put into 3ml LB, added 3l Strep and put in a shaking incubator.
We also made competent cells.

Week 4

We ligated full construct g-block into Psb1C3 plasmid backbone.
Diluted primers to 100mM, made set of 5mM primer tubes. Transformed ligated plasmid into top 10 cells (still some plasmid left in the top shelf of the freezer). PCR: 5l 5mM forward primer
5l 5mM reverse primer
0.5l of gblock
25l of Q5 mastermix
14.5l of water
The ligated plasmid transformed cells did not grow overnight so were repeated. We undertook PCR for zf10, zf14 and zf2

Week 5

Both of the chemically transformed plates and electrotransformed plate grew over weekend.
LppompAzif268: colony PCR of clone 2 ok (inoculate culture to purify DNA inoculate 2 cultures, 4.5ml each. Miniprep them after making a glycerol stock).
Conducted plasmid miniprep of bacterial culture. Gel electrophoresis of redone PCR Pgsa worked, zf2 (all others need to be repeated at a later stage).
Running gel of miniprep restriction digestion. Redoing PCR: INP, zf2, zf14 (67oC). zf10 (69oC) with DMSO (0%, 3%, 6%).
PCR purification of BCLA and PGSA (nanodrop returned negative, redoing). Redoing PCR of INP, zf2, zf10, zf14.
Redid PCR of INP, zf10 and zf14. zf14 returned positive, purified (31.8 ng/l. Redoing PCR of INP and zf10. Purified zf2, nanodropped.
Gel of zf10 and INP returned negative.
Redoing PCR with 66oC and 680C annealing temperatures.
Restriction digestion protocol: 5l cutsmart 10x 1l DNA 10 units enzyme each make up to 50l water
For Nde1-Age1-HF, incubate at 370C for 5-15 minutes.
Diluted INP g-block and ZF10 g-block by a factor of 10.
Ran gradient PCR of INP and ZF10 (made 12 tubes of 20l).

Week 6

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Week 7

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Week 8

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Week 9

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Week 10

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Week 11

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Week 12

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Final Week

This is the content of the last section

Sep 21

@@ Line 130: / Line 130: @@
 <br>Conducted plasmid miniprep of bacterial culture.  Gel electrophoresis of redone PCR  Pgsa worked, zf2 (all others need to be repeated at
-a later stage).  Running gel of miniprep restriction digestion.  Redoing PCR: INP, zf2, zf14 (67oC). zf10 (69oC)  with DMSO (0%, 3%, 6%).  PCR purification of BCLA and PGSA (nanodrop returned negative, redoing).  Redoing PCR of INP, zf2, zf10, zf14.
+a later stage).  <br>Running gel of miniprep restriction digestion.  Redoing PCR: INP, zf2, zf14 (67oC). zf10 (69oC)  with DMSO (0%, 3%, 6%). <br> PCR purification of BCLA and PGSA (nanodrop returned negative, redoing).  Redoing PCR of INP, zf2, zf10, zf14.
 <br>Redid PCR of INP, zf10 and zf14.  zf14 returned positive, purified (31.8 ng/l.  Redoing PCR of INP and zf10.  Purified zf2, nanodropped.
+<br>Gel of zf10 and INP returned negative.
+<br>Redoing PCR with 66oC and 680C annealing temperatures.
+<br>Restriction digestion protocol: 5l cutsmart 10x
+l DNA
+units enzyme each
+                                                            make up to 50l water
+<br>For Nde1-Age1-HF, incubate at 370C for 5-15 minutes.
+<br>Diluted INP g-block and ZF10 g-block by a factor of 10.
+<br>Ran gradient PCR of INP and ZF10 (made 12 tubes of 20l).
 </p>
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Difference between revisions of "Team:Warwick/Project5"

Revision as of 12:25, 19 August 2015

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