2 replies to this topic

[et2b]

Hi everybody,

Has anybody any tips to help us cranck up our read lenght when bisulfite sequencing on a ABI?
Our read lengths are currently a dismal 160 bp, when we need up to 300 bp read length (useable read length).

Does anybody have some tips to increase the trace length?

Thanks a bundle in advance!!!
ET2b

[methylnick]

et2b,

are you direct sequencing or cloning and sequencing?

I will assume you are direct sequencing as we have had no problems with cloning and sequencing, the primer that you use to sequence could be a little lousy, you could try increasing the number of cycles in the sequencing reaction (up to 40-50) or throw in a little bit of Q solution (from qiagen) this seems to improve things in our hands.

Another thing you could try is to tail one of your primers with T7 and sequence using the T7 primer.

good luck

Nick

[moonlitleaves]

Hi,

Do anyone can provide me any protocol to do sequencing for disulfate samples? I always failed to get long reads from the results. I had added betaine and dGTP and some there are some background noise also.

It will be great if anyone can help.

thanks.
moonlitleaves