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#1 biocrazy

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Posted 04 December 2010 - 03:36 AM

Dear Friends,

I prepared competent cell by CaCl2 method. But it seems the competency is very weak (i.e I get less than 10 colonies in the transformation). What can I do to improve the competence? Could anyone have this experience. Thanks in advance!

#2 pDNA

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Posted 04 December 2010 - 04:04 AM

First of all calculate the competency of your cells ...this gives you a ratio on the used DNA and the cfu's you get. This is an important step if you want to judge if your protocol for the generation of cells works fine or if you messed it up. 10 colonies could be much ...or nothing ...always depends on the initial amout of pDNA you used!

Another thing that is important during preperation of CaCl2 cells:
Cells should be handled with care. No vortexing or excess pipetting should be performed, specially when the cells have been resuspended in CaCl2 because they tend to be fragile, decreasing the viability and therefore the competency.

There are millions of protocols out there in the WWW ...go and ask google. You will see that nearly every lab does it somehow different. I normally stick do Sambrook&Russel when starting with a new protocol ...and if i dislike a certain step i go out and search for different protocols or ask other people how they do it.
...and to be honest ...i do not use CaCl2 competent cells anymore ...for my lab electroporation works best.

Regards,
p

#3 christy

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Posted 06 December 2010 - 04:27 AM

Hi,

take the log phase cells (0.5 - 0.8 OD cells), centrifuge should be around 5000 rpm. pellet resuspension in calcium chloride should be very slow. it will take hours together only on ice.




Dear Friends,

I prepared competent cell by CaCl2 method. But it seems the competency is very weak (i.e I get less than 10 colonies in the transformation). What can I do to improve the competence? Could anyone have this experience. Thanks in advance!






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