oligo splint for ssDNA

Steven Russell Genetics sr120 at mbfs.bio.cam.ac.uk
Sat Feb 5 05:57:57 EST 1994


Bill writes

>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"
 5'-XXXXXXXXXXXXXXXXXXXXXXXXXXX5'3'YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY- 3'
               3' OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 5'
                        Oligonucleotide "splint"


>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.

Steve Russell
Dept of Genetics
University of Cambridge
UK

SR120 at mbfs.bio.cam.ac.uk





More information about the Methods mailing list