Protocols

Genetic Engineering

Whole Genome Isolation

• 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

Generating electrocompetent cells

• 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

Transformation via electroporation

• 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