self-ligation of small dsDNAs
robert at arbo.microbiol.uwa.oz.au
Tue Jul 7 00:21:35 EST 1992
>2. I have no idea because the only way most of us do this expt is to use
> some means of biological selection (i.e. circularize a linearized plasmid)
> and after transformation, grow in selection media (i.e. antibiotic) and
> only those cells expressing the phenotype (i.e. antibiotic resistance)
> will grow because the harbor the plasmid.
The above fragment is a small part from Bruce Roe's reply. There is another
way to test whether an oligo has autoligated. Use for example an oligo (ds)
with Eco ends. Demonstrate you can visualise it with fill reactions using
hot A together with Klenow (15 min on the bench). To minimise
concatemerisation use a low concentration of oligo in your ligation (use
Maniatis as a guide). Test whether you can fill. If the bulk of your oligo
has ligated you should see nothing on a gel (so have a marker !!), if there
was a certain amount of concatemerisation you'll see fragments which are
larger than the one-mer.
I know this is not an answer to the original question, but it does provide a
method for assessing what happens in a ligation reaction.
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