upper limit to Inverse PCR products?

Brad Nicholson Brad_Nicholson at hlthsci.med.utah.edu
Fri Jan 19 12:52:24 EST 1996


In article <broekh-1801962142350001 at port43.annex2.net.ubc.ca>, 
broekh at unixg.ubc.ca (Henry Broekhuyse) wrote:
>snip<
 >For those of you with more time/interest to brainstorm, here's what I 
did:
>more details snipped>

Hi Henry,

I have a couple of ideas, but first the caveats.  I work with Salmonella, 
so my IPCR is a lot less complex, and DNA is easier to come by.  

1)  To address the intermolecular (good) vs. intramolecular (bad) problem, 
try ligating a 1:10 dilution of a restriction mix.  If you normally use 10 
ul (from the restriction digest) in your ligation, try 1 ul.  You may be 
tying up your DNA ends with other fragments instead of making the oh so 
wonderful circles.

2)  Could you try a 4 cutter?  Maybe something that is rare amongst 4 
cutters, but more common than the 6 cutters that aren't working.  You could
be really unlucky and be entering a region without a lot of decent sites, 
which can make your life miserable.  

These are things that I have used before, it may or may not be usefull for 
you.

Good luck,
Brad 

===========================================================================
Brad Nicholson                      |"If it worked the first time, 
Department of Pathology             |	it wouldn't be research."
University of Utah                  |	 ...Brad Nicholson
Salt Lake City, UT 84132            |  My opinions are solely my own.
Brad_Nicholson at hlthsci.med.utah.edu |
or: (801)-581-4365                  |  iligitimi non corborundrum
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