Annealing ssDNA oligos. Is it easy?
jhartley at access2.digex.net
Thu Oct 12 06:30:50 EST 1995
It is easy and reassuring to follow this on a high percent agarose gel.
Apply 10 pmol of each oligo, and 10 pmol of your hybrid, to a 3 or 4%
agarose gel. Each single stranded oligo will migrate according to its
base composition and any secondary structure, and will stain poorly with
ethidium. The duplex will give a big bright band, and migrate
considerably more slowly in the gel. This also tells you if the
concentrations of the oligos are about right, since excess of either will
be visible below the duplex. I use a minigel format run pretty warm
(about 7-8 v/cm) with a fan for cooling, to keep the bands tight. Only
takes 20 minutes to run.
On 10 Oct 1995, Joaquim Culi wrote:
> Dear netters,
> I don't know how to do in order to obtain an efficient annealing of two ss
> complementary oligos. They are around 50 mer. After annealing I want to
> ligate them as a monomer and also as several concatamers, so they have 5'
> and 3' overhanging BamHI-BglII sites.
> I will appreciate any protocol or reference.
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