Team:UNIPV-Pavia/Calendar/June/settimana5

June, 28th

First time in wet lab

11 LB agar plates, 35 LB agar + Amp plates and 15 LB agar + Cm 12.5 were prepared.

• BBa_C0060 was resuspended from iGEM 2011 kit distribution, Plate 1, well 4K in 15 ul of ddH2O
• BBa_C0061 was resuspended from iGEM 2011 kit distribution, Plate 1, well 4M in 15 ul of ddH2O
• BBa_J04450 was resuspended from iGEM 2011 kit distribution, Plate 1, well 5E in 15 ul of ddH2O
• BBa_K081022 was resuspended from iGEM 2011 kit distribution, Plate 2, well 12N in 15 ul of ddH2O

For each of them 1 ul was transformed in 100 ul of home-made TOP10 competent cells. Transformants were plated on LB agar plates with the right antibiotic and incubated ON at 37°C.

June, 29th

In the morning all plates showed colony; for each one we picked one colony and inoculated it in 1 ml of liquid LB with the proper antibiotic (ampicillin for BBa_C0060, BBa_C0061 and BBa_K081022, kanamycin for BBa_J04450). Cultures were grown at 37°C, 220 rpm.

In the afternoon glycerol stock were prepared (750 ul of bacteria and 250 ul of 80% glycerol); then cultures were refilled with liquid LB with the proper antibiotic and incubated ON at 37°C, 220 rpm.

June, 30th

PhaP-1 and PhaP-2 plates showed colonies!! A colony from each plate was picked and used to infect 1ml LB+Kan. Liquid cultures of PhaP-1 and PhaP-2 were grown for 6 hours at 37°C 220 rpm and then used to prepare glycerol stocks. Remaining liquid cultures were re-filled with 5 ml LB+Kan an grown ON at 37°C 220 rpm for tomorrow MiniPrep. Glycerol stocks of PhaP-1 and PhaP-2 are stored at -80°C in the iGEM 2010 Registry box.

Ligations were heated at 65°C for 5 minutes to inactivate ligase ad then tranformed in E. coli TOP10 home made competent cells. Plates were incubated ON at 37°C (we decided to incubate plates 5 hours longer than usual, because cell growth was already slower for I6).