Circularization and ligation of ssDNA
tk at mit.edu
Wed Jul 5 08:08:55 EST 2006
"rijuta kotenkar" <rijutak at googlemail.com> writes:
> Could anyone tell me a protocol for circularization of ssDNA using a
> complimentary helper oligo which binds at the ligating end.
> I tried many protocols but failed to get quantifiable detection of
> circularized molecules.
> I am using T4 ligase.
I believe the standard approach is to use T4 RNA ligase (note: not
DNA ligase). This ligates ssDNA (and RNA) effectively without any
helper oligos. Check the protocols for inverse PCR, which often
involve a circularization step prior to PCR. Also be aware that
the NEB buffer shipped with T4 RNA ligase has no ATP, which is
required. If you use the buffer shipped with T4 DNA ligase instead,
it already has ATP added.
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