cloning problems

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Jul 12 11:29:22 EST 2002


When you say you "sequenced" it, do you mean you sequenced the PCR 
product before you cloned it?

If that's the case, it's not surprising to find individual clones 
with mutations.  When you sequence a PCR product, you are sequencing 
a population.  Any one mutation will not show up, because it only 
accounts for a (small) fraction of the population.  However, once you 
are working with a cloned PCR product, individual mutations are 
obvious.  I recently cloned an approx 1.2 kb PCR product.  I used 
limited cycles, but conventional (non-proofreading) Taq.  Of the six 
clones I sequenced, one had no mutations, while the other 5 each had 
at least one mutation.  No two mutations were the same.

To minimize the number of mutant clones, I'd suggest using as few 
cycles as possible and perhaps use a proofreading polymerase mix. 
Sometimes too, it might be possible to swap pieces from clones to 
make one that is free of mutations.

Hope this helps.

Mike

>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


-- 

Michael L. Sullivan, 
Ph.D                                                            
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax
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