McGill/Team 2: Repressilator
From 2007.igem.org
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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):
- Amp: 1uL/mL
- Kan: 10uL/mL
- 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
Jh0001
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.
- I-brick from yesterday's midi prep
- I-brick from yesterday's midid prep (2)
- RSEI
- RSE2
- pEGFP mini prepped
- pTet mini prepped
- Elvis's pGFP
The gel from yesterday's midi prep was successful and DNA was present.
Dilutions where implemented on the seedings from yesterday
June 2007
June 4
- Performed a restriction digest of midi preps: pEGFP, pTet, J brick. pEGFP (EcoRI & XbaI); PTet (Xba1); J brick (EcoRV & Pst)
- After digest, made a gel using L2 Ladder. To prepare L2 use 3:1:1 (H20:Dye:TAE).
Wells: 1. pEGFP 2. pTet 3. J Brick - Seeded the following plates: I+J (20ul&100ul); I1500S (1x with LB & 1x with M.M.); J4000I (1x with LB & 1x with M.M.)
Gel showed abnormal bands with smudges. This could be due to improper well loading or gel transfer.
June 5
After seeding, results showed that the J brick grew abnormally, growing better with M.M. than with LB. I+J (double seedings) did not grow at all in both mediums. The I brick grew in LB but not with M.M.
Conclusion: Possibly bad colonies were selected or there were problems with the transformation procedure.
- Transformed plates again along with Supplemental plates.
- Seeded I, J and I+J (2007 & 2006 plates) again, 3 colonies selected each as well as 2 Control tubes (1x LB; 1x LB+Kan/AMP)
June 6
- Made 23 Kan+Amp plates.
- Transformed commercial biobricks (I15004, J40001, Elowitz (I5610)) in pSB1A2 plasmid (AmpR) with Stble 3 cells. Left overnight in incubation room.
June 7
Results from last night's transformation:
I15004
20ul: 3 big colonies, many small colonies
100ul: 20 big colonies, many small colonies
J40001
20ul: 2 big colonies, few small colonies
100ul: 7 big colonies, no small colonies
I5610
20ul: 1-2 small colonies, little growth
100ul: 1-2 small colonies, little growth
Seeded plates with LB. 1 colony from each plate chosen (2x each biobrick). 6 seedings in total. Left in S/I overnight.
June 8
Of last night's seedings, only two tubes showed visible growth: I15004(100ul)& J40001(100ul). No growth seen in I15610(Elowitz) in both concentrations.
Performed a Miniprep of I15004 & J40001. Kept in freezer.
June 11
Restriction Digest and Screening: I15004 and Jh0001 plasmids miniprepped on Friday were digested as per the following
I15004
1.5 uL Buffer 3
0.5uL BSA
4uL DNA
7uL water
1uL PVUII
1uL PstI
J40001
1.5 uL Buffer 3
0.5uL BSA
4uL DNA
7uL water
1uL EcoRV
1uL PstI
Digestion was run on gel though there were no bands as the TAE was improperly made. Seeding were repeated to be prepared tomorrow (2X I15004 20uL, 2X I15004 100ul, 2X Jh0001 100uL).
Transformation: I15610 was transformed into commercial STBLE 3 cells with a HS of 45s and plated onto AMP resistent plates.
