How to prevent melting of short DNA duplexes
S.H at l.u.c
Fri Mar 27 10:36:53 EST 1998
In article <3517F8B5.1AA2 at chugaibio.com>, dspinella at chugaibio.com,
> Dear BioNetters:
> Does anyone know of a simple way to enhance the stability of short
> double-stranded (say 10-12 base pairs) DNA fragments in a complex
> mixture? I'm trying to find a way to prevent even AT-rich sequences of
> this size from melting apart in solution that won't interfere with
> subsequent enzymatic manipulation of the fragments (particularly
> ligation). Suppose for example you were trying to make a library of 12
> bp fragments. How could you clone the fragments into a vector under
> conditions that wouldn't select against the AT-rich fragments because
> they melted apart before encountering the vector? Obviously, I could
> increase the salt concentration, and/or decrease the temperature of the
> solution (with attendant adverse affects on the subsequent ligation
> reaction). How about TMAC? Polyamines? Any suggestions would be
> appreciated. Thanks.
> --Dom Spinella
I had exactly (well, more or less) the same problem a few days ago. I
solved it by first phosphorylating the oligos (T4 PNK) then assuming that
the single-stranded linkers would ligate first to the double stranded
vector, and only then would they anneal for a final ligation.
E.g: 8 pb phosphorylated linker, with an EcorV site:
5'-XXXXXXXXXXXX + 5'-XXGATATC
CTATAGXX-5' + XXXXXXXXX-5'
1h at RT
O/N at 16°C
And there you are!
The only important thing, is that your oligos need to be previously
phosphorylated, otherwise it will never work that way...
I hope it helped
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