[Q] How to anneal long (695b) heteroduplex DNA fragments?

Peter Rudolph Peter.Rudolph at MPI-Dortmund.MPG.de
Mon Jan 20 04:35:07 EST 1997


Hi,

we want to create heteroduplex DNA by annealing the WT with a single-base 
mutant. Both fragments are double-stranded DNA cutted out of a vector and gel 
purified.

The only problem is, that the fragments are 695bp long. We simply tried to 
pipet them together in a buffer containing 0.67M NaCl, heat them up (5min in a 
boiling water bath) and anneal them by slowly lowering the temperature - but 
all we got was a very large DNA fragment (multimer?) which didn't even run 
into a 4.5% non-denat PAG. We could detect only a very small amount of DNA in 
the expected size. Even the adding of formamide in concentrations up to 60% 
didn't help at all - in fact, we got even more of the big multimer and almost 
nothing in our expected size. 

So has anybody done a comparable experiment before? And how can one calculate 
the Tm of a fragment that big - is the %GC-formula still valid for that size? 
Would it be better to make single-strand DNA first and anneal them afterwards? 
Does the addition of formamide, DMSO or SSB help at all?

Any help is highly appreciated!!

Thanks in advance,

Peter.



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