Adela.Torres at uv.es (Adela Torres Calatayud) wrote:
>>I'm currently working on DNA non-radioactive cycle sequencing, and I'd like to
>know if there is a reliable formula by which I can choose the annealing
>temperature of the PCR program when I'm sequencing using custom-made oligos.
>Right now the formula I'm using is:
>> 2(A+T) + 4(G+C) - 6 = T ann.
>>But it didn't give me good results when I tried it. So if there's anyone out
>there that can help me, thanks very much! E-mail or post.
>> Adela Torres
> (Adela.Torres at uv.es)
The formula 2(A+T)+4(G+C)=Tm is OK for oligos less than 20-mers and at 1M salt,
with annealing at 5-10° under Tm. For sequencing or PCR, salt concentrations
are much less. For larger oligos or for taking into account salt concentration,
I use the formula 81.5° + 16.6log(base10)M +o.41(%G+C) - 820/n, where M is the salt
concentration in molar and n is the length of the oligo. In both editions of Maniatis,
the formula is given as -16.6logM and 600/n. The first part is obviously wrong. U
nder 1M, the log becomes negative, and Tm diminishes with salt concentration. A
s for the length part, during site directed mutagenesis when I used differential
hybridisation to distinguish between mutants and wild type, I found that the f
ormula I use is good to about 2°. However, I do all the hybridisations ot 6SSPE, w
hich is about 1M, so logM resolves to zero. If anyone has any good information a
bout the validity of formulae at lower salt concentrations, i.e. about 0.1M for P
CR, I would be glad to hear about it.