Team:Carnegie Mellon/Week2

Monday June 10th:
- Met with Natasa about modeling
- Made 0.5 L LB Agar and 0.5 L LB broth
- Autoclaved
- Added chloramphenicol to the agar
- Poured plates (remember to put plates in fridge!)
- Looked through registry for Lac promoters
- Transformed 3 promoters (BBa_R0011, BBa_K864400, BBa_R0010) and killer red into E. coli
- BBa_R0010 and killer red on ampicillin, other two on chloramphenicol

Promoters
http://parts.igem.org/Part:BBa_R0011 plate 3, 5C (inconsistent) (chloramphenicol) -lambda pL hybrid
http://parts.igem.org/Part:BBa_K864400 plate 1, 17H (confirmed) (chloramphenicol) -ptac/trp/lac
http://parts.igem.org/Part:BBa_R0010 plate 5, 1D (confirmed) (Ampicillin) -WT lac promotor

Tuesday June 11th:
Colonies on chloramphenicol plates had non-uniform size
possible that chloramphenicol was rather old
Left LB/cam plates out all night :(
plated with remaining samples of BBa_R0011, BBa_K864400, BBa_R0010 to see if plates are still ok
Ordered promoters and lambda arms/ packaging extract
We inoculated 4 cell cultures and stored them in 37 degree room
Cheryl is developing primers for cloning

Wednesday, June 12
Plates with remaining samples of BBa_R0011, BBa_K864400, BBa_R0010 confirmed that plates are ok. Plate with strain that was only amp resistant had no colonies, two plates with chloramphenicol resistant plasmids had individual colonies, not a bacterial lawn (yay!)
Mini-preps for all four plasmids
Ran 2 uL DNA on gel- lanes right to left- standard; 5C lambda pL; 1D WT lac; 17H ptac/trp/lac; killer red

Poor yield of 1D lac believed to be caused by pipetting error
Multiple bands in KillerRed lane probably because plasmid is large and was nicked during prep

Thursday, June 13 PCR of KillerRed and Primers
Gel of PCR products:
Top row (left to right): ladder, Killer Red x3 with EcoKRF and EcoKRstR primers, eGFP x3 with EcoEGFPfor and EcoEGFPstR primers, Killer Red x3 with SpeRBSKRF and PstStKRrev primers
Bottom row (left to right): first two are Cheryls, no DNA controls x4 (For: KillerRed1, eGFP, KillerRed2, promoters from left to right), pTac/trp/lac, WT lac, lambda pl, ladder (SpeRBSKRF, PstStKRrev primers for promoters)

Friday, June 14
repeated PCR with KillerRed and primers SpeRBSKRF and PstStKRrev
primers were long and were at room temp for several minutes before cycler started, so we think they formed primer dimers based on gel from June 13
made gel: 0.4g agarose, 40ml TE buffer, swirl to break up agarose. microwave 1 min 30s in erlenmeyer
Swirl again to prevent agarose settling and add 2 \micro\ liters ethidium bromide and swirl to mix
Pour gel in taped mold
Decided to clone KillerRed into plasmid (high copy?) first because phage parts won’t be here until next Wednesday
Sent ptac promoter DNA (H17) and WT EColi lac promoter to be sequenced (s/b here by tues)
5 μL H_2O, 5 μL DNA, 1 μL 10 μM primer (VF2)
iGEM1: ptac promoter
iGEM2: WT E. Coli lac promoter
From left to right: ladder, KillerRed template with primers SpeRBSKRF and PstStKRrev x2, control and KillerRed with primers as first 2 rows (whoops) and control