oligo splint for ssDNA
Steven Russell Genetics
sr120 at mbfs.bio.cam.ac.uk
Sat Feb 5 05:57:57 EST 1994
>I'd like to hear from anyone who has experience using an oligonucleotide
>as a "splint" to allow ligation of two single stranded DNA ends.
>What I mean is:
DNA molecule "X" DNA molecule "Y"
3' OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 5'
>Questions I have include the minimum regions of complimentarity needed, the
>efficiency of the reaction, ligation conditions and the tolerence for base
>mismatches between the oligo and the DNA.
>Any help or suggestion most appreciated.
I tried some of these experiments 5 or 6 years ago so me memory's a bit hazy.
I think I was using a 30mer oligo (~15 bp complementarity each side) and
ligating overnight at 15oC with T4 DNA ligase. As far as I remember there was
an NAR paper published on a cDNA cloning method which used this technique
around 1988-89, I've just looked for the ref but cant find it. I remember we
designed the oligo such that one side had a much higher Tm than the other so
that we couls anneal, dilute and circularise. I have no info on missmatch
but should be standard oligo missmatch reduction in Tm.
Dept of Genetics
University of Cambridge
SR120 at mbfs.bio.cam.ac.uk
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