Asymetric PCR

John Rebers jrebers at nmu.edu
Mon Dec 18 16:51:21 EST 1995


A technician working with me got some odd results when setting up an
asymetric PCR reaction.  The template is purified phage DNA, with an insert
of Manduca sexta (moth) DNA of about 14 kb.  When an equimolar amount of
primers about 500 nucleotides apart are used, a band of the expected size is
seen on a native agarose gel, along with a minor band of about 1200
nucleotides.  When the 2 primers are at a ratio of 5 pm: 50 pm, the minor
band is significantly stronger, but still weaker than the 500 nucleotide
product.  With a ratio of 50 pm: 5 pm of the same primers are used, 4 minor
bands appear, all weaker than the major product.  The gel was stained with
SYBR Green II.

Are the slow-migrating bands likely to be an artifact due to the presence of
ss and ds DNA in the same gel, or is this more likely to be a problem with
non-specific binding of the primers giving rise to several amplification
products from the phage template?  I'm inclined to sequence the amplified ss
DNA, and see if it gives readable sequence that matches that expected for
this region.

Any possible explanations or hints to avoid the slow mystery bands would be
appreciated.
John Rebers
Department of Biology
1401 Presque Isle Avenues
Northern Michigan University
Marquette, MI  49855
906-227-1585 (office)
906-228-3617 (home)
906-227-2013 (FAX)
jrebers at nmu.edu




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