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Small competent cells


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#1 Janina

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Posted 30 November 2004 - 03:47 AM

The competent E. coli cells I produced grow very slow. I tested DH5 alpha and Nova Blue, both grow slower than before.
I grow an overnight culture in 2 ml LB, then inocculate 1 ml in 100 ml LB + 10 mM KCl + 20 mM MgSO4 and grow them to an OD600 = 0.5. After this, I cool them down on ice for 10 min, centrifugate 5 min at 2800 g, 4 C, resuspend pellet in 40 ml buffer I ( 30 mM Potassiumacectate, 50 mM MnCl2, 10 mM CaCl2, 100 mM RbCl, pH 5.8) and incubate again on ice for 10 min. Next steps are: centrifugating 5 min, 2800 g 4 C, resuspending in 4 ml buffer II (10 mM MOPS, 75 mM CaCl2, 10 mM RbCl, 15 % v/v glycerol, pH 6.5) incubating 10 min on ice, freezing in pre-cooled tubes.

Now I don't understand why the stains, treated by same procedure, result in smaller colonies.
Any suggestions? :unsure:

Thanks

#2 Charon

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Posted 30 November 2004 - 04:45 AM

Well, it is normal cells that were exposed to cold-shock grow slower.
You'll see this for insance with glycerine cultures.
During the adverse conditions the metabolic activity will be strongly reduced and it takes a while before they recover.
Or did you mean that the alphas are growing slower than the novas?

#3 ravibiot

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Posted 30 November 2004 - 07:24 AM

hi
well, i too had the same problem that you are facing now...
i substituted CaCl2 based competent cell preparation method (Gene cloning Maniatis, 1982) instead of RbCl2 based method and observed good results.
i hope that you too can try with the same. Also, you can check the LB medium age (use fresh plates), use appropriate final concentrations of selection pressure (antibiotic) and check the incubation temparature.

Best regards,
Ravi
INDIA

#4 Janina

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Posted 01 December 2004 - 12:37 AM

I compared efficiency of "old" and "fesh" competent cells, both for alpha and nova. And both fresh cells grow slower... This method seems to work, but the cells have to grow more than 16 hrs to result in a good size for picking.

Well, I'll try Maniatis method next time, I guess. :unsure:




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