Andy Law (Big Nose)
lawa at bbsrc.ac.uk
Tue Sep 6 14:26:59 EST 1994
In article <199409020012.RAA17831 at netcomsv.netcom.com>,
jloring at genpharm.com ("Jeanne Loring") wrote:
> Hello netters- I followed some of the recent thread about the problems with
> cloning PCR fragments, and, since I now have the problem, I wish I'd listened
> more carefully. I cut my fragment with the restriction enzymes whose
> believed I had included (with a 5'clamp), but there was no way to be sure they
> both worked (SacI and XhoI). Now I'm not getting inserts into
Bluescript II. A
> colleague is having a similar problem with an entirely different sequence and
> plasmid. Was there a summary on the net about solutions? I did read that
> treatment of the Taq-produced fragment with the Klenow fragment of DNA
> polymerase solved the problem for some people. Has anyone else had luck with
> that, or can you suggest another solution?
Basically IMHO there are two possible solutions that have worked for me.
1) Forget about the fancy restriction-sites-in-my-primers method.
Sacrifice directional cloning and use a T-tailed vector to directly clone
the PCR product.
2) Digest the PCR product with Proteinase K PRIOR to restriction digestion
(Crowe et al., NAR 19)
Doing both of the above i.e. digesting with PK prior to direct ligation
into a T-tailed vector covers all eventualities. I found that 1) alone was
successful nearly 100% of the time.
If you do want the direction restriction site method, then remember to
include at least 3 bases (in my experience) hanging over the end of the
restriction site. Primers with sites right at the end of their sequence DO
NOT WORK. If your primers are designed like this (with the restriction
site right at the end) then don't panic. Just clone it into a T-tailed
vector, grow it up, cut it out of the plasmid and then clone it in the
direction you want.
( Lawa @ bbsrc.ac.uk Big Nose in Edinburgh )
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