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Inverse PCR (or alternative) recommendations requested

J. David Spafford jspaffor at gpu2.srv.ualberta.ca
Thu Nov 30 11:24:47 EST 1995


In Article <1995Nov29.210119.12773 at alw.nih.gov>, bernard at elsie.nci.nih.gov
(Bernard Murray) wrote:
>Hello all,
>        I am considering using inverse PCR to attempt to locate transgenes
>in the mouse genome.  Before starting I thought I would pick your
>collective brains on the topic.  Is this the technique of choice or has
>it been superceded?  Can you use long range PCR to extend well into
>the flanks?  What's the longest fragment that has been amplified?.  Can
>conventionally purified genomic DNA be used or do special precautions
>have to be taken?
>        I have read two book chapters and several original articles
>on the subject but was wondering how things work out at the practical
>level.  Any advice/warnings/pointers gratefully received.
>                Bernard
>
>Bernard Murray, Ph.D.
>bernard at elsie.nci.nih.gov  (National Cancer Institute, NIH, Bethesda MD, USA)
>

I amplified a 1100 bp genomic fragment using inverse PCR.  Too much template
and all I could see were smears.  Check concentrations of template over a
1000 fold range for the PCR.

Good luck,





J. David Spafford,
Department of Biological Sciences, University of Alberta
jspaffor at gpu.srv.ualberta.ca



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