cloning problems

Warren Gallin wgallin at gpu.srv.ualberta.ca
Fri Jul 12 11:33:20 EST 2002


Daren,

	Sequencing the uncloned PCR product will always give you the correct
sequence because the individual mutations that are caused by infidelity
in the PCR reaction are each only a minor component of the whole
mixture.  However, if the reaction is run under conditions in which the
PCR reaction is not optimally faithful, then a lot of and sometimes most
of, the individual molecules will have a mistake or two.

	The solution is to run the PCR reactions a little differently.  One
factor is to try lower dNTP concentrations, that will help decrease
incorrect incorporation.  Another factor is to try to find the smallest
number of cycles you can run and still get a usable amount of DNA.  The
third thing is to use a proofreading enzyme, or spike a Taq reaction
with about 1/10 to 1/5 of a proofreading enzyme, for any PCR reaction
that you will be cloning from.

	Those are just the three things that we have found to eliminate the
problem you outline.  There are likely many more.

Warren Gallin

Daren Rice wrote:
> 
> I am having trouble subcloning a cDNA our lab is interested in.  I used
> PCR to generate the cDNA and then sequenced it to confirm there are no
> errors in the sequence.  When I clone it using DH5a bacteria, the
> resulting plasmid I get back has mutations in the cDNA sequence. Each
> individual clone I pick has different mutations from the others.  The
> mutations are mainly point mutations that change a single amino acids,
> but sometimes I get single base deletions or insertions that shift the
> reading frame, and once I even had a rearrangement.  I have tried other
> bacterial strains such as GM33- and M1H, but am still having no luck.
> We have just ordered Stratagene's SURE bacterial strain which has some
> mutations in genes involved in DNA repair and recombination that should
> make them less susceptible to changing my DNA.  If anybody has any
> tricks for cloning difficult DNA, I would greatly appreciate them.
> 
> Thanks,
> Daren Rice
> drice at kumc.edu



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