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Bisulfite sequencing is giving problem - (Sep/17/2010 )

Hi,
I was doing direct bisulfite sequencing and have a problem in reading chromatograms.

First I amplified a clean target sequence using bisulfite sequencing primer. Purified and did the sequencing reaction using Bigdye.... Again purified the sequencing reaction, dried and dissolved in formamide and forwarded to ABI 3730 sequencing.

Initially,it reads very good (up to 60-90bp). It gives a very good and clean peaks but after 60-90bp, the peaks drop all of sudden, which can't be read.

I want to know why its coming like that........

any one can help?????/

DK

-dkmishra-

Polymerase is probably hitting a homopolymer or is trying to go through it and falls off. Are there a run of A's or T's at that position? To solve it you would need to come in from the other side.

-methylnick-

No, I cannot see the homopolymer (run of A's/ T's) before it falls off.

I used both Forward and reverse primers and it happening in both cases.

DK

-dkmishra-

Try adding additional "cold" (i.e. unlabeled) DNTPs to your sequencing reaction. I routinely dilute my DNTP-Stock (25ÁM each) 1:1000 and add 1Ál to my sequencing reaction (carried out in 10Ál total, also using ABI BigDye 1.1). It should enhance your reading length significantly.

Good luck!

-Timmay-

Timmay on Wed Sep 22 16:00:27 2010 said:


Try adding additional "cold" (i.e. unlabeled) DNTPs to your sequencing reaction. I routinely dilute my DNTP-Stock (25ÁM each) 1:1000 and add 1Ál to my sequencing reaction (carried out in 10Ál total, also using ABI BigDye 1.1). It should enhance your reading length significantly.

Good luck!


Thank you Timmay.
I will do it today.

DK

-dkmishra-

Does sound like you potentially have your template/primer ratio wrong therefore you have premature terminaion.

-methylnick-

methylnick on Fri Sep 24 21:39:52 2010 said:


Does sound like you potentially have your template/primer ratio wrong therefore you have premature terminaion.


I am using the standard concentration of template/primer concentration.
Template (ng)= 9*xkb for 1 ul
Primer = 10pmole each rx (10ul)

-dkmishra-

Which version u r using Big dye V1.1 or 3.1?

-sarada-