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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.
Jun 29
7/7/15
We made 8 plates of streptomycin and chloramphenicol 3 plates of chloramphenicol Cells made electrocompetent following lab protocol
8/7/15
Cells grew!
Cells transformed-
INP 8H psB1c3 5
Ipp OmpA 6L psB1c4 5
Zif 23 1G psB1c5 3
9/7/15
Met Miriam phD presentation and explanation of cloning/ gibson assembly/pcr
4 minipreps Nanodrop results: J04450 55.1ng/l
INP 10.8 ng/l
C2001 16.1 ng/l
LPPOMPA 38.4 ng/l Electrophoresis: Made 10% urea, 1% agarose gel ethidum gel
Put plasmids from mini prep in gel, ran for 30 mins at 100V
(Left to right: Ladder, LPPOMPA, C2001, INP, J04450)
13/7/15
Grow up MG1655 (Z1) cells. Put into 3 ml LB
Added 3l Strep
Put into shaking incubator (37oC) overnight.
14/7/15
Made competent cells (followed competent cells protocol)
22/7/15
Ligated full construct gblock into Psb1C3 plasmid backbone.
23/7/15
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
24/7/15
Ligated plasmid transformed cells did not grow overnight, repeating today.
PCRing the bcla, inp and pgsa.
Transformed more cells with ligated plasmid using electroporation, plated.
Also PCRd zf10, zf14 and zf2.
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).
3/8/15
Redoing restriction digest of LPP-OMPA
Running gel from Friday (last time = 6.1 ng/l, issue with plasmid?)
Plasmid concentration only 5.9ng/l, running ligation anyway.
20l ligation reaction (full details in book).
4/8/15
Transforming ligated plasmid into mg1655 cells.
PCR purify INP.
Restriction digestion of INP and LPP-OMPA.
Diluted full construct g-block 10x.
Gradient PCR of LPP-OMPA full construct.
5/5/15
2 colonies from a grown DH5 Z1 plate were selected, 2 cultures inoculated, grown in shaking incubator.
Purify and restriction digest full construct.
INP digestion.
Streaked plate with PGSA and BCLA transformed cells.
6/8/15
ZF10 gel electrophoresis.
Cut out ZF10 (at 270 bases, according to ladder).
Gel extraction (freeze ‘n’ squeeze).
7/8/15
Streaked chlor plates with transformed RFP DH5 Z1 cells.
Re-streaked plates with BCLA and PGSA transformed cells.
Made chemically competent DH5 Z1 cells (frozen at -80oC).
vRan gel of digested plasmid, only obtained single band at 2000 base pairs (where backbone without RFP would be, lacking band at 1000 base pairs (for RFP gene)).
Ran gel of undigested plasmids, single band at 3000 base pairs.
Gel purified digested plasmid, nanodropped (2.1ng/l).
8/8/15
RFP and BCLA transformed cells grow with single colonies.
PGSA plate showed no growth.
Inoculated falcon tubes with RFP and BCLA. Also inoculated tube using old PGSA, incubating in shaking incubator.
Plated pure DH5 Z1 on a chlor and a clean plate (to establish background growth).
9/8/15
Miniprep of RFP, BCLA and PGSA transformed cells.
Chlor control plate grew no colonies, clean control plate grew a lawn.
10/8/15
Nanodropped miniprep of RFP, BCLA and PGSA.
Sent off for sequencing.
11/8/15
Digested old EC1 plasmid using Pst1, EcoR1 and Age1, Nde1 to obtain plasmid backbone and full construct … anchor proteins.
Digest new RFP plasmid to obtain plasmid backbone.
Inoculated culture with RFP bacteria, need to mini-prep tomorrow.
PCR if sZF10.
Mini-prep of RFP plasmid (JO4450).
12/8/15
Prep of 2 binding slides (overnight shaking 37oC, 6 hours 60oC oven, turned halfway) and 1 control (HCL bath only).
Miniprep new RFP plasmid, digest, gelled.
Gel of old full construct digested by Age1 and Pst1, gel extracted, nanodropped.
Gel extraction didn’t work.
Digestion of all zinc fingers.
13/8/15
Ligation of lpp-full construct into PSB1C3 backbone.
Transformation of ligation product.
14/8/15
Restriction digestion of full construct using Pst1 and EcoR1.
Inoculation of full construct transformed plasmid.
Only 2 colonies on the 4 plates of bacteria grew, background growth is 0 via control plates.
15/8/15
Mini-prepped grown transformed DH5 Z1, colonies 1 and 2.
Plated normal DH5 Z1 on clean plates, found lawn, viable cells.
Re-transformed cells formed a lawn, taken to be re-spread, will inoculate tomorrow.
17/8/15
Prepared miniprep for sequencing.
Digested the lpp out of the plasmid and gel extracted the backbone.
Ligated all anchor proteins into the plasmid.
18/8/15
Transformed DH5 Z1 cells with each anchor protein.
Re-growing lpp transformed DH5 Z1 cells, plan to make glycerol stock later.
Electroporated MG1655Z1 cells using the original full construct plasmid.
Plated/streaked FIM(negative) cells on a clean plate.
19/8/15
All plated grew well, lpp transformed cells formed lawn.
Streaked lpp transformed cells.
Electroporated cells did not grow.
Inoculations were made of the BCLA, PGSA and INP plates.
Made 12 new chlor plates.
Designed primers for the modellers.
Sequences came back with no mutations!
Colony PCR of all grown cells, will run on gel tomorrow.
Aug 24
Aug 31
Sep 07
Sep 14