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 colonies (BBa_J04450 showed light red colonies); 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 stocks were prepared (750 ul of bacteria and 250 ul of 80% glycerol) and stored at -80°C except for BBa_C0061 which was not sufficiently grown (we let it grow until next morning); then cultures were refilled with 5 ml of liquid LB with the proper antibiotic and incubated ON at 37°C, 220 rpm.

June, 30th

All cultures were saturated; glycerol stock for BBa_C0061 was prepared and stored at -80°C.

Plasmids were extracted with MiniPrep kit; purified DNA was quantified with NanoDrop Spectrophotometer:

BBa_B0030, BBa_B0031, BBa_B0032, BBa_B0034, BBa_B0015, pSB4C5, BBa_I13501, BBa_I13507 were inoculated from freezer glycerol stocks and grown ON at 37°C, 220 rpm for MiniPrep.