Strange problems with Invitrogen TOPO vectors Reply-2

Earl Blewett blewett at osuunx.ucc.okstate.edu
Tue Oct 5 16:58:56 EST 1999



Greg Hawkins wrote:

> Has anyone had problems with TOPO vectors (especially the TA cloning
> vectors)?  We sequence these clones for customers and have found restriction
> sites that flank the cloning site missing, sequencing primers that do not
> work on one side of the cloning site, plus other strange things we can not
> explain.  Is the topoisomerase causing so strange recombination that screws
> up the regions flanking the cloning site?

Sorry about  the first message, text seems to have disapeared.

We  have had the same problems. I have tried to clone 4 products ranging from
1800-3600 and my tech has tried a 3600 bp piece. I got lot's of colonies and
when minipreps were done they were all the same thing. Not the right size, not
the right restriction sites present and when sent to be sequenced to find out
just what it was none of the sequencing primers would work.

We use rTth for the amplification, gel purify the product, add A residues with
Taq and wizard-prep it. The final product is a clean band when run on a gel.

I talked to Invitrogen last week about the problems and they suggested reducing
the incubation time after the insert DNA is added to the Topo vector to nothing.
Just add the DNA and immediately transform the bacterial cells.

We did this on the weekend using their controls as well as one of our products.
Their control worked ours gave 3 colonies, none were the desired clone.

We are repeating it this week with a different product.

My DNA is 60% GC, an orf, no inverted repeats or unusual secondary structure, no
long poly-nucleotide stretches. I know, I've sequenced it. I'm cloning alleles
for sequencing and phylogenetic comparison.

If anyone has any ideas I'd be glad to hear about them.

Earl Blewett Ph.D.
Biochemistry and Microbiology
Oklahoma State University-College of Osteopathic Medicine
Tulsa, OK

blewett at osuunx.ucc.okstate.edu




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