Gene synthesis using oligos

Rafael Maldonado 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 
machine.

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.


Rafa



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