PCR from genomic dna

tweissen at my-deja.com tweissen at my-deja.com
Thu Jun 15 06:09:52 EST 2000


Here are some possible reasons that just came rattling
off my head:

- Genomic DNA offers has more sites for mispriming,
leading to short fragments (sometimes confused with
primer dimers) which outcompete your target. You should
see them as a fuzzy band at the bottom of the gel,
usually 30 - 70 bp: Adjust stringency.

- Genomic DNA is more refractory to denaturation, as
mentioned by previous posters: Try predenaturation
(alkaline or heat), shearing, or restriction digestion.

- Genomic DNA preps are more likely to be contaminated
by salt or SDS, if not done carefully: repreciptiate
(but don't loose it all :-) ).

- Genomic amplicon contains large intron: redesign primers.


I don't think Mg++ concentrations will drop more with
genomic than with plasmid template. Any other offers?

Thomas

Thomas Weissensteiner
Edward Jenner Institute for Vaccine Research
Compton RG20 7NN, U.K.
Fax: 0044 1635 577901
http://www.horizonpress.com/gateway/pcr.html


In article <392EE3F3.9C4C5096 at tribe.ulm.edu>,
  vaidyaks at tribe.ulm.edu (Kedar Vaidya) wrote:
> I have been trying to get a specific part of the dna amplifies by
> PCR..the primers seem pretty ok. they work when i pcr from plasmids
but
> i get no product from the genomic dna...any suggestions?
>
> --
>
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> Kedar S. Vaidya
> Graduate Student in Pharmacology
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> 700 University Avenue
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