Competent DH5 alpha
Posted 16 March 2009 - 03:31 AM
When I do transformation with my competent cells I don't have so much colonies.
Who can help me?
Posted 16 March 2009 - 04:36 AM
Posted 16 March 2009 - 07:12 AM
waiting Your e-mail (and your protocols),
How many samples do you need? I can offer you one protocoll for about 60 and one for about 250.... Greets
Posted 17 March 2009 - 04:03 AM
Preparing electro competent cells
Two methods were used in order to get different volumes of competent cells:
1) small volume (~15 tubes)
Inoculate 2ml ONC in 100ml LB medium plus antibiotics (1:50)
Incubate at 37°C till an OD¬550 of 0.5-0.7
Divide into two 50ml Sarsteds tubes (precooled) and leave 15-30 min on ice.
Centrifuge in a table top centrifuge for 7min at 4500rpm (3645xg) and 4°C
Discard supernatant (completely!) and carefully resuspend pellets in a total volume of 50ml 10% Glycerol (cold!)
Centrifuge like above
Discard supernatant (completely!) and carefully resuspend pellets in altogether 25ml 10% Glycerol (cold!)
Centrifuge like above
Discard supernatant (completely!) and carefully resuspend pellets in 0,5ml 10% Glycerol. Make aliquots.
The cells can now be kept cool till usage or shock frozen in liquid nitrogen and kept at
2) big volume (~250 tubes)
Inoculate 1l of LB with 1/100 volume of fresh ONC plus antibiotics
Grow cells at 37°C with vigorous shaking to an OD600 of 0.5-0.7
To harvest, chill the flask on ice for 15-30 min, and centrifuge at 4000g and 4°C for 15min.
Remove as much of the supernatant as possible. It is better to sacrifice the yield by pouring off a few cells than to leave any supernatant behind. Resuspend pellets in a total of 1l ice-cold sterile ddH2O taking care not to lyse them. Centrifuge as above.
Resuspend in 0.5l of ice-cold sterile ddH2O. Centrifuge as above.
Resupend in ~20ml of ice-cold sterile ddH2O. Centrifuge as above.
Resuspend to a final volume of 2-3ml in ice-cold sterile 10% glycerol. The cell concentration should be about 1-3x1010 cells/ml.
This suspension may be frozen in aliquots on dry ice and stored at -70°C. The cells are good for at least 6 months under these conditions.
Posted 17 March 2009 - 06:29 AM