"
Team:Bielefeld-Germany/Labjournal/Molecular
From 2013.igem.org
m |
m |
||
| Line 30: | Line 30: | ||
<style type="text/css"> | <style type="text/css"> | ||
.iso_whole{display:none;} | .iso_whole{display:none;} | ||
| - | . | + | .eccs{display:none;} |
| - | . | + | .e_trafo{display:none;} |
</style> | </style> | ||
Revision as of 21:53, 25 September 2013
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
