Team:UNIPV-Pavia/Calendar/August/settimana2

AUGUST: WEEK 2

August, 9th

Phasins plates showed very few colonies (12 for I20-new and 17 for I21-new): they were all picked and let grow in LB+Amp 100ug/ml. In the evening we made glycerol stocks and re-filled the tubes for the screening of the following day:

In order not to loose our time we decided to perform again the PCR-amplification/modification of <partinfo>BBa_K208001</partinfo> in case of a negative screening. Than we gel-ran and gel-extracted right amplicons.

Check of LB+Cm 6 ug/ml agar plates. We let grow at 30°C:

• MG1655 in LB
• MC1061 in LB
• MG123/008 in LB+Amp 50 ug/ml
• MC123/008 in LB+Amp 50 ug/ml

Than we streaked cultures on a six-sector divided plate, and we let grow it ON at 30°C. Since 6 ug/ml is a very low concentration, we wanted to check if actually nothing (without the right resistance) grow on these plates.

Transformation at 30°C of 100 ul of MG13/008 and MC123/008 competent cells to check their efficiency. We used 1 ul (~4 ng) of RING, F2620-4C5, Nothing (water) and plated on proper LB agar plates:

• RING: Cm 34 ug/ml
• F2620-4C5 (positive control): Cm 12,5 ug/ml
• Nothing (negative control): Cm 12,5 ug/ml

Both RING and F2620-4C5 should survive, but we had a problem so that MC008 transformed with RING couldn't be plated. We will check it another time. We let grow plates ON, 30°C.

August, 10th

August, 11th

August, 12th

August, 13th

August, 14th

August, 15th