Gene synthesis using oligos
rafael at corona.med.utah.edu
Fri Apr 12 15:54:05 EST 1996
On Thu, 11 Apr 1996, Dr. Duncan Clark wrote:
> Hi Folks,
> I need to synthesise the first 130bp of a gene. Rather than try to using
> two v.large oligos I thought of using four smaller overlapping ones and
> then PCR'ing using the two outers or using klenow to fill in the gaps.
> My question is how many bases should I overlap on each oligo? Is a
> homolgy of ten bases at each end of an oligo enough?
Yes, it is. Even 8 bases is used in some mutagenesis protocols. But for
so small fragment, I would order only two oligos; counting the
overlapping region, they will be 70 b long, good enough for a nice oligo
By the way, may places can make a 130 bases oligo, so you don't have to
extend at all; just order two complementary oligos, aneal them and clone.
Also, I use klenow and clone directly the product. With the regular
amounts of oligo you get, it is more than enough. Just remember that
Klenow (and other enzymes) lacks the 5'-3' exonuclease: you will get
nick at the begining of an internal oligo. If you are going to clone, it
is fine; but if you are going to PCR that, it won't work.
Rafael Maldonado | 'No te creas todo
room 6160 Eccles Institute of Human Genetics | lo que leas en
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