Team:Groningen/Labwork/12 July 2013

Mirjam

Made a PCR with (1) no strep tag (2) strep F (3) strep R. The annealing temperature for (1) and (2) is 78 degrees Celsius. For (3) the annealing temperature is 80 degrees Celsius. Gel purification of the strep F silk obtained yesterday.
Run a 0.8% agarose gel for 30 min at 90V. Made a gel for the PCR products. Four out of six revealed bands at the expected height. Made a new ligation between digested munchen backbone and the LacI promoter. Check of the restriction and ligation of the signal sequences and the silk. No bands appeared on gel.

Chaline

Gel purification of the silk PCR products.

Chaline and Mirjam

Run a gel with the ligation product and the purified silk fragments.
Silk is present for all three kinds, although in a low concentration.

Inne

Made 3 bottles of 250 ml LB Agar (20g/L LB, 16g/L Agar)
Autoclaved:
3 bottles of demiwater (80 ml)
3 bottles of tabwater (80 ml)
3 bottles of milliQ (80 ml)
3 bottles of LB Agar (250 ml)
1 bottle of milliQ (1 L) Ran a gel with silk ligation at 100V for 22 min

Sander and Claudio

made antibiotic stock.
Ampicillin 50mg/ml MQ water
Chloramphenicol 5mg/ml Ethanol 50%
Kanamycin 10mg/ml MQ water
Spectinomycin 10mg/ml MQ water
All stocks are filter sterilized and all except Chloramphenicol are stored at 4 degrees. Chloramphenicol is stored at -20 degrees