Q: Can you random prime a 250bp PCR product?

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Thu Jul 30 10:30:46 EST 1998


At 11:43 7/30/98 GMT, Makoto Kamei wrote:
>Hi all,
>
>I am about to screen a mouse genomic DNA library constructed in EMBL3
>lambda vector. I have ~250bp PCR product made from mouse genomic DNA
>template. Is it possible to use this product as a probe for the screening?
>Is it possible to random prime this product to make a probe? Any help
>appreciated. Thanks a lot in advance.

Yes, absolutely.

I had to screen a genomic southern for copy number and had to use single 5'
and 3' exon probes for both mouse and human C5 - both very large genes.  

The 5' probe was 110 bp.  The key:  1) a labeling system that works well (
we have used either Promega and BMB's sytems, and  2) the freshest
radiation possible.    This is far too small for nick-translation - the
smallest I've done was 600 bp and it worked but was no where near as good
as a 3 kb n_translated probe.

Don't expect 10^9 cpm/ug, due to the size.  As long as you get 10^8 cpm/ug
it should work.

Hope this helps,
David
=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
Never take life seriously. Nobody gets out alive anyway.
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