McGill/Team 2: Repressilator

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May 2007

May 11

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)

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 PGFP (which was supposed to be 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 incubator 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 plates (200µl & 50µl) and RSE with Amp plates (100µl & 20µl). Left in incubator overnight at 37C.

Also, six seeding were preformed on test colonies transfected with PGFP and let in the IS overnight.

May 24

Miniprep protocol (from iGEM 2006) of Top10 and STBLE3 cells transformed with pGFP plasmids and seeded yesterday. BL21 cells were tested for the presence of the pGFP plasmid through microscope examination which proved positive results.

A restriction digest was implemented on the isolated DNA from the Top10 and STBLE3 cells 1.5uL Buffer 2
0.5uL of BSA
3uL DNA
8uL water
1uL EcoRI
1uL XbaI

Due to lack of ethidium bromide, a gel could not be made so the run was postponed for Friday.

May 25

First, the Top10 and STBLE3 were again digested as per the digestion above to be run in parallel with the digestion from yesterday.

An electrophoresis gel was made 50mL of TAE
0.35g agarose
heated ~20s then cooled to <60 degrees Celsius
7uL of ethidium bromide was added and poured into a gel plate

1.74uL of 10X loading dye was added to the DNA and the gel was run for 45 minutes at 100V; upon completion, a picture was taken to reveal no bands.

A miniprep was also preformed on the iBrick (Il5004 plasmid) and RSE cells (J40001 plasmid) seeded the day before and DNA stored in the freezer.

May 28

Today, a restriction digest was preformed on the iBrick and RSE cells with the following respective solutions in duplicate

IL5004
1.5uL Buffer 3
0.5uL BSA
3uL DNA
1uL PvuII
1uL PstI
8uL water

J40001
1.5uL Buffer 3
0.5uL BSA
3uL DNA
1uL EcoRV
1uL PstI
8uL water

Again, a gel was run and again, a negative result was obtained showing no bands.

May 30

Midi prep of iBrick with a new midi prep kit.

Seeding of J-brick (RSE) and Jay's pEGFP and pTet

May 31

A gel was run with the following wells.

1. I-brick from yesterday's midi prep
2. I-brick from yesterday's midid prep (2)
3. RSEI
4. RSE2
5. pEGFP mini prepped
6. pTet mini prepped
7. Elvis's pGFP

The gel from yesterday's midi prep was successful and DNA was present.