HELP! Bacterial strains for PCR cloning

Rafael Maldonado rafael at corona.med.utah.edu
Tue May 9 14:13:58 EST 1995


On 9 May 1995, Michael Cooley wrote:

> sl6dp at cc.usu.edu wrote:
> : I have been trying to clone PCR fragments with no success. My primers
> : have restriction sites, the enzimes seem to be cutting and the ligation
> : seems to be working fine. I am using JM110 E. coli strain, and someone
> : suggested that this may be the problem because this strain does not
> : have the "hsd" mutation, and the bacterial endonucleases will chew-up
> : my cloned PCR product because it is not metilated. Does anyone heard of
> : this proble with certain strains for cloning PCR-amplified DNA. I am
> : surprised that I've never found a word about it before????
> 
> Your friend is correct. JM110 would not be suitable. Besides the lack of 
> an hsdR mutation, it has no endA mutation which is another endonuclease 
> you can do without. This is not however restricted to cloning PCR 
> products. Later, Mike

If you use a restriction plus strain in your PCR cloning, you'll get a 
lower fequency of transformants, but not a blank. Anyway, it's better 
using a hsdR mutation (r- m+); I would use a recA strain too; JM110 is 
recA+ and this can cause problems if your DNA is unstable because 
toxicity, some repeats or homology to the chromosomal DNA. The endA mutation 
won't affect your transformation eficiency, but the yield in plasmid extraction.

So... why are you still using JM110? You can use DH5a, XL1-Blue or many 
other more.

But you should get some transformants, anyway. I suppose you have checked 
the restrictions, the compentent cells, the ligation (BTW, how do you know 
that your ligations are working properly?) and that the PCR product is the
right one!

Rafa

___________________________________________________________________
                                             |
Rafael Maldonado                             |  "y es que...	  
room 6160 Eccles Institute of Human Genetics |   habemos gente pa to'"
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