Designing methylation primers on long CpG island - (Mar/21/2005 )

Hi again!

I now also got bands with methylnick's primers (both pairs).

Any advice for the sequencing, except those already given by pcrman?


klahar

Woohoo it's all happening Klahar!!!

Pcrman's advice is all good. It is more challenging to directly seqeunce the amplicon, although it would give you are very idea of the methylation status of the population of amplicons.

You could clone into a sequencing vector. This is an easier method, though you have to seqeunce quite a few clones (at least 8) to give an *estimate* of the population.

Direct sequencing will only cost one reaction (more if you have to optimise primers). Cloning and then seqeuncing costs more time (and more money) although you will be sure that the seqeuncing reaction will work because you will use universal primers to seqeunce.

your choice! flip a coin!

Nick

Hi klahar,

In your case, I think cloning would be better and it will give you very clean data. Direct seqeuncing often has noisy background especially if there is no much methylation.

Just one more question. Does anyone know any polymerase with similar efficiency as JumpStart RedTaq from Sigma, but which is cheaper?
My boss thinks it's too expensive to buy a lot of it.

/klahar

We routinely use promega master mix or amplitaq for bisulfite sequencing with great success. It think Sigma offer the jump start at a very competitve rate, here in australia that is.

Nick