upper limit to Inverse PCR products?
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:
>For those of you with more time/interest to brainstorm, here's what I
>more details snipped>
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
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
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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