McGill/Team 2: Repressilator

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Revision as of 21:16, 23 May 2007

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May 2007

May 11

1. Autoclaved LB, Agar and Minimal Medium. Made plates with AmpR, KanR and both Amp and Kan. Concentrations used (same for cell cultures):
   1. Amp: 1uL/mL
   2. Kan: 10uL/mL
   3. Cam: 0.8uL/mL (from Elvis' aliquots)
2. The receipe for M9 Minimal Medium is in my lab notebook which is in the lab so I'll post it later under the Protocols section. Hopefully we can get the 2007 wiki soon. Horia

May 14

Moved plates from iGEM fridge to fridge on C block (end of hall) Made 100X Yeast extract and 10X Dextrose (for supplementing minimal medium) Prepared CaCl2 and CaCl2/Glycerol 10% solutions for CC procedure of tomorrow Will seed tonight. Horia

May 15

Performed cc procedure with top10 (elvis' home made), bl21 a1 and stble3

Performed seeding procedure on plates from last year: ILS004, duplicate copies of 5mL LB with 50uL KAN; RSE/J40001, duplicate copies of 5mL LB with 5uL AMP; Place in IS overnight

May 16

Transformed cc cells with pUC19 and plated

Repeated seeding procedure as that from previous day did not prove any results (defective shaker?). Both ILS004 and RSE repeated in triplicate with its respective antibiotic. Placed in IS at noon and two samples diluted with LB (100mL to RSE sample and 80mL to ILS004) and placed in the IS overnight. No cell growth.

May 17

Nothing grew on most of the plates. Only 2 colonies on a stble3 plate. Decided the cells were bad because 1. we did not keep them on ice while we transported them etc. and 2. some cells waited a long time on ice because we had to do sequential centrifugations (not enough of one kind of tube). Removed from incubator at night and placed in the fridge.

Susan, Avi and Jimmy seeded cells again for Friday. BL21, Top10 and STBLE3 repeated in triplicate in 5mL LB and placed in SI overnight.

May 18

Performed cc procedure.

May 22

Made LB for Seeding procedure from 15.5g of solid LB to 500ml of water - x3.

Performed transformation of competent cells with pUC19 (Top 10 and Stble 3) and BL21 with PGFP and Amp antibiotic to validate their quality.

For the transformation procedure, used LB instead of S.O.C. Medium, due to its unavailability, then spread the cells on Agar gel plates with Amp and LB and placed in SI overnight.

Made some additional Agar gel plates with Amp, which were kept in the freezer for future use.

May 23

Test transformation results

BL21
30s / 2 min Heat Shock
20 µl = 0 cells / many cells
75 µl = 0 cells / many cells

Top 10
30s / 2 min Heat Shock
20 µl = many cells / many cells
75 µl = many cells / many cells

Stable 3
30s / 2 min Heat Shock
20 µl = ~ 30 cells / 7 cells
75 µl = ~ 30 cells / 3 cells

Then performed transformation using Top 10 cells. Added biobrick (10M well) with Kan - (200µl & 50µl) and RSE with Amp (100µl & 20µl). Left in incubator overnight at 37C.

June 2007