brett at BORCIM.WUSTL.EDU
Sun Feb 11 11:35:51 EST 1996
>chung006 at mc.duke.edu (Namjin Chung) writes:
>>I have been experiencing very frustrating subcloning blues. I have PCR'd
>>1-kb DNA fragment with PstI restriction site and extra 2-bp at each end.
>>Then, the fragment was agarose gel-purified, digested fully (I tried one-
>>and two-overnights), and gel-purified again. I tried ligation of this
>>fragment to a vector of 7.3 kb which was completely cut with PstI and
>>dephosphorylated. I should have positive clones, but got nothing - there
>>was even no antisense ligation!
>>According to NEB, PstI-site-containing fragment with extra 2-bp's should be
>>cut over 90% after an overnight. However, is there any possibility that my
>>fragment was not cut and I hasseled around with this uncut fragment? I
>In the lab I work in, we have done extensive restriction site introduction
>using PCR. From a variety of anecdotal sources, it seems that cloning of PCR
>products by this method is often difficult, and we've had problems as well.
>The way we currently deal with this is by designing extra long primers, with
>an additional *6* bases after the restriction site, rather than the NEB
>suggested number of bases after the site. (Actually, I go overboard with a
>full *9* extra bases...)
>In your situation, with only 2 bases after the PstI site, if you don't want
>to make new primers, I would recommend TA cloning the crude PCR product and
>then cutting out of the TA vector with PstI... (Sure beats trying to blunt
>the PCR product in, because end-polishing has been VERY unsuccessful in my
>hands for PCR products.)
Ouch, more work! I suggest polishing the ends (if needed), as Joe suggested.
Multimerize your PCR reaction via blunt end ligation, and recover your fragment
by a PstI digestion. I suggest following aliquots on agarose. This simple
procedure often allows for better ends in cloning.
Program in Immunology
Washington University - St Louis
brett at borcim.wustl.edu
"I own my own pet virus. I get to pet and name her." - Cobain
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