Bisulfite sequencing is giving problem (View forum version)



dkmishra

Posted 17 September 2010 - 12:35 PM

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

methylnick

Posted 17 September 2010 - 01:48 PM

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.

dkmishra

Posted 20 September 2010 - 09:15 AM

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

Timmay

Posted 22 September 2010 - 08:00 AM

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

Good luck!

dkmishra

Posted 23 September 2010 - 07:42 AM

Try adding additional "cold" (i.e. unlabeled) DNTPs to your sequencing reaction. I routinely dilute my DNTP-Stock (25M each) 1:1000 and add 1l to my sequencing reaction (carried out in 10l 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

methylnick

Posted 24 September 2010 - 01:39 PM

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

dkmishra

Posted 27 September 2010 - 07:27 AM

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)

sarada

Posted 13 October 2010 - 11:00 PM

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