ligating single stranded DNA
gene_jockey26 at yahoo.com
Mon Apr 24 17:33:25 EST 2000
How efficient is in vivo ligation of ds or ssDNA. In some of our cloning
procedures where we want to shuffle an insert between two backbones we mix
both plasmids, cut with the appropriate enzymes, ligate, then counter-cut
with an enzyme whose site is only present in the original backbone. This
(linearizes) eliminates religation of the original donor clone and ligation
of backbone one to backone two. Both our donor and recipient backbone are
Km-r but the donor is also Ap-r. When we screen Km-r colonies for
Ap-resistance after counter-cutting, they are always Ap-sensitive. So is it
possible that a particular strain is required for in vivo ligation, if it
does occur? We use JM107 for routine cloning.
"Jim Kami" <jakami at ucdavis.edu> wrote in message
news:3904808E.719B4DA2 at ucdavis.edu...
> Not only is it possible but very practical for a number of novel
> cloning procedures. The "Band-Aid" oligo just needs to be annealed to
> the fragment and transformed. I've been told that the ligase step is not
> really necessary as the host cell will do that for you. A nice
> application of this method is to use small oligos to create restriction
> sites. The enzyme will only cut at the ds DNA region and then use a "
> Band-Aid" oligo to religate the plasmid. A nice method when there are no
> unique restriction sites in your region of interest. The only reference
> I can think of offhand is one by Arrow and Dale 1985. I think it was in
> Methods in Enzymology, but it's been so long I can't be sure.
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