Sequencing a stretch of Ts - (Jul/30/2008 )

Hi
I am trying to sequence a clone which has about 20 ish Ts in a row. Does anyone have any tricks ie primer design etc. that could potentially help us know accurately the number of Ts? thanks, Cindy

-cindyb-

It's very difficult. Strand slippage is a problem, both in the sequencing reaction and in the PCR of the template before that.
I'd suggest that you sequence from both ends to estimate the number of T's. You can also prime to that region with an anchored primer TTTTTTTTTTTTTTTTTTVN and sequence out from that region. Lowering the PCR and sequencingextension temperature down from 60 will also help. Breathing of the primed sequence is a problem with high AT regions.

-phage434-

I concur with phage434. In addition to the suggestion made, another thing to do would be to limit the number of cycles of the reaction. PCR always favours the smallest product. And with the strong possibility of slippage, this will be a particular problem.

Emulsion PCR has been suggested to be able to overcome this problem (Nature methods).

-perneseblue-

QUOTE (phage434 @ Jul 30 2008, 06:46 PM)

Thank you very much for your help-- yeah, its hard to know whether I have the right thing and the sequence is a bit off (by a T) or like you said the PCR could be off in the first place. I like the anchored primer idea and was thinking that may be a good go-around. Thanks again- Take care, Cindy

-cindyb-

QUOTE (perneseblue @ Jul 30 2008, 07:13 PM)

Thanks Perneseblue-- if I do go with the anchored primer-- I will lower my cycle number-- makes sense. Youve been very helpful
Take care, Cindy

-cindyb-