Primer synthesis problems

Austin P. So (Hae Jin) nobody at
Mon Dec 6 15:04:10 EST 2004

Paul Shinn wrote:

> To the other poster, I would love to cartridge purify these
> oligos but I am doing hundreds and it is cost prohibitive.

I guess it is a matter of cost versus results, no? Aren't they like $5 
bucks pop?

> We do an overnight digest with the Sfi because we've found that
> anything less than 8 hours is insufficient and gives us poor
> transformants. are under the assumption that your SfiI site is intact in the 
  amplification product...

Along the lines of Duncan Clark's suggestion, depending on the sequence 
of your oligo at the 5'-end, and depending on the polymerase you are 
using, editing might be occurring during PCR. I'm assuming that you are 
using a proof-reading polymerase since you appear to want to maintain 
the SfiI site. Like I said (and I'm starting to lean towards this 
possibility), your SfiI site can lead to the formation of a hairpin, and 
depending on the loop sequence of the hairpin as well as the bases 
flanking the loop, you can have hairpins that melt at >70C (note that 
this means that at the Tm, 50% of the oligos will be should 
look up papers by Benight on some unusual hairpin thermodynamics). A 
proof-reading polymerase will deal with this hairpin in any number of 
ways, which may explain your product distribution.

Either directly cloning your PCR product (TA cloning) or sequencing the 
5'-ends of your PCR product (say by using a primer within the oligo 
since it is big enough) before cloning may be able to resolve the source 
of your problems.


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