11mer hybridization problem

Austin P. So (Hae-Jin) nobody at nowhere.com
Tue Oct 18 17:31:23 EST 2005


vagos wrote:
> Hi everyone, I would appreciate some help on this one:
> I am trying to anneal two single stranded 11mers that have a base
> mismatch. The sequences of the top and bottom strands are:
> 
> TCATCAACTTC
>     GCTGAAGCTTC
> 
> I want this ds11mer to be ligated to a ds55mer.
> 
> However, the I see no 66mer when I analyse the ligation product in a 4%
> agarose gel. Instead, I can clearly see the 66mer band when I use the
> ds11mer that has no base mismatch:
> 
> TCATCGACTTC
>     GCTGAAGCTTC
> 
> I assume that the mismatched oligos do not hybridize at all. Do you
> have any suggestions on how to overcome this problem?
> Thank you in advance,

It is really not an 11-mer hybridization...

Although I think it is unlikely to work anyway...I would say to perform 
the ligation in 20% PEG-8000 final (make a 50% w/w solution in water as 
a stock) and see what happens.

However, you may also get spurious ligation/concatenation products, so 
it may be better to titrate the amount of PEG-8000 to find an optimum 
concentration.

cheers

Austin


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