direct digestion of colony PCR product

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Fri Apr 8 04:25:15 EST 2011


Dear Marta,

basically, I'd just try it. Simplifying lab procedures is always a
good idea.

You might check the buffer conditions (salt concentration, pH,
magnesium conc) that your enzymes require. If they don't work (well)
in your PCR-buffer, you might consider adding sort of homemade
"differential" buffer(s) for each enzyme and possibly do a sequential
digest (starting with the low salt requiring enzyme). Possibly the
contribution of PCR buffer is small, so diluting the reaction a bit
with water and adding 10x digestion buffer could make sense. If your
template DNA interferes, you may cut it into small chunks with DpnI
(suppose it's methylated, when you use "standard" E. coli).

In worst case, just precipitate your PCR reaction with isopropanol,
wash with 70% EtOH (RT ist good, no need for any freezing), dry (you
may do a third wash with ether to remove residual EtOH and water to
accelerate drying:).

If you need to do some downstream cloning, beware that Pol and
nucleotides still are present in your mix, so you'll end up with
blunted/added overhangs/chewed fragments dependent on your Pol. In
that case, use PCR cleanup columns that positively catch your PCR
product. Adding one µl of phenol saturated water to the PCR reaction
is good for the paranoid to kill (any) remains of Pol.

Have fun!

Wo


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