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.
Read more Jun 29
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.
Week 6
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Read more Aug 3
Week 7
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Read more Aug 10
Week 8
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Read more Aug 17
Week 9
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Read more Aug 24
Week 10
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Read more Aug 31
Week 11
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Read more Sep 07
Week 12
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Read more Sep 14
Final Week
This is the content of the last section
Sep 21