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Competent DH5 alpha


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#1 FRAROAR

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Posted 16 March 2009 - 03:31 AM

Hi, I'm searching a good protocol to have wonderful competent cells..
When I do transformation with my competent cells I don't have so much colonies.
Who can help me?

#2 isbow

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Posted 16 March 2009 - 04:36 AM

How many samples do you need? I can offer you one protocoll for about 60 and one for about 250.... Greets

#3 FRAROAR

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Posted 16 March 2009 - 07:12 AM

Thank You, I'm happy for Your e-mail. as i've said You before, when I trasform my competents I obtain few, few colonies. So it's important for me to learn a new method to prepare competents.
waiting Your e-mail (and your protocols),
Regards,


Francesco



How many samples do you need? I can offer you one protocoll for about 60 and one for about 250.... Greets



#4 isbow

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Posted 17 March 2009 - 04:03 AM

Hi! These are the two methods I used to make competent cells in my diploma thesis. Important is to really keep them cold and you can also let them stand for half an hour or more before you shockfreeze them, a collegue told me that the cells like that. With these protocolls they should be highly competent.... good luck

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
-70°C.

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.

#5 memo

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Posted 17 March 2009 - 06:29 AM

Use TRAEN enhancer in your transformations (my home-made competent transfprmation were giving about 100 cfu's extra (normally max 10 cfu))




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