Team:Bielefeld-Germany/Labjournal/Molecular

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Genetic enginering protocols



For the isolation following kit has been used: Fermentas GeneJET™ Plasmid Miniprep Kit

- Centrifuge 10 mL of over-night liquid culture

- Resuspend pellet in 800 µL of resuspension solution

- Ribolyse three times for 60 s with 6500 rpm (30 s break between every run, 400 mg beads)

- Centrifuge 3 min at 10,000 rpm

- Transfer 500 µL of supernatant into new 2 mL tube

- Add 500 µL of lysis buffer, invert 4 - 6 times

- Add 700 µL of neutralisation buffer, invert 4 - 6 times

- Centrifuge 10 min at 12,000 rpm

- Transfer 2 x 600 µL of supernatant into spin column (centrifuge down each time for 30 s at 12,000 rpm)

- Two wash steps with 750 µL of washing buffer

- Dry column

- Elute in 75 µL of elution buffer



Material

- 550 mL LB-Medium

- 1 L cooled bidest. H2O

- 150 mL cooled 10 % glycerine

- 10 pre-cooled 50 mL Falcons


Protocol:

- Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm

- Inoculate 2x250 mL LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm

- Incubate until OD600 0,4-0,6

- Cool the culture 15-30 minutes on ice

Onwards all steps at 4°C

- Divide the cultures into cooled 50 mL Falcons and centrifugate at 4000 rpm, 4 °C for 15 minutes, make sure to slowly accelerate and deccelerate

- Discard supernatant

- Resuspend pellet in 5 mL cooled bidest H2O (and don't get frustrated while doing it, keep shaking gently)

- Pool two suspensions each, add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)

- Discard supernatant

- Resuspend pellet in 5 mL cooled bidest H2O

- Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)

- Discard supernatant

- Resuspend pellet in 5 mL cooled 10 % glycerine

- Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above)

- Discard supernatant

- Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend

- Divide cells in 100 μL aliquots and freeze in liquid N2 immediately

- Store at -80 °C



- Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary

- Add 0.5-5 µL plasmid to 50 µl electrocompetent cells

- Store cells on ice for 1 minute

- Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ώ

- Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C

- Centrifuge 2 min at 2000 rpm and plate on selective LB-Medium

- Incubate over night at 37 °C