ss DNA from pcr?

Basavaraju Shankarappa bsh at MED.PITT.EDU
Mon Feb 8 09:46:55 EST 1993


> hello there,
> this is a repost as i didnt recieve any replies last time.  my system was down
> and i do not know if any one replied. if you replied to this question last time
> it was posted, could you please reply again, as i got no posts.  thanks in
> advance.
> I am looking for a pcr protocol to generate, or otherwise obtain large amounts
> of single stranded cDNA.  i have the m13 method, but i would prefer to use a pcr
> method if anyone has any good ideas.  i hope some of you people out there may
> have some ideas for me.....thanks in advance.......
> rod
> Rod Bonfiglioli, Waite Agricultural Research Institute,
> University of Adelaide, South Australia.
> e-mail rbonfigl at waite.adelaide.edu.au

	I can draw your attention to three following publications which
deal with the production of ssDNA for either sequencing or mutagenesis.
In my experience, the efficiency of production of ssDNA by PCR is quite low.
This becomes logarithmically (?) exasperated as your size increases.
It may do some good to look at these publications.

Gyllenstein and Erlich.  Generation of ssDNA by PCR and its application to
direct sequencing of HLA-DQA locus.  PNAS (1988) 85: 7652

McCabe, P.C. 1990.  Production of ssDNA by asymmetric PCR p76-83 in
Innis et al ed., PCR Protocols, A guide to methods and applications
Academic Press, London.

Perrin and Gilliland 1990.  Site-specific mutagenesis using asymmetric PCR and
a single mutant primer.  Nucl Acid Res. 18: 7433-7438.

Anyway, Good luck
Raj Shankarappa
Univ of Pittsburgh
bsh at med.pitt.edu




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