Q: Sequencing GC rich regions // Looking for sequencing kit/method using a LABELLED TERMINATOR (ddNTP)

Wolfgang Schechinger diabetes at lrz.uni-muenchen.de
Mon Sep 2 16:30:09 EST 1996


Hi folks! Sorry for the long subject.

Actually I wanted to use a kit from Amersham with 33P radiolabelled
ddNTPs to overcome my sequencing problem: unspecific terminations
-staggering enzyme- in a GC rich sequence. Unfortunately my lab
doesn't have the permit to use 33P yet. Thus I'm looking for hints on
other kits or methods to overcome this problem. Yet I have tried 
* cycle sequencing from plasmid DNA with 7-deaza GTP
* "convential" plasmid sequencing (dITP, dGTP)
* lowering the termination temperature from 37C to 30C to prevent the
polymerase from slipping off the DNA
* running a 40% formamide gel

but always I get these unspecific terminations.

Now I want to try another way of radiolabelling the sequencing
products by using radioactive ddNTPs in order to see only the
correctly terminated DNA transcripts on the gel. But not using 33P.
32P or 35S is OK. (Maybe we're kind of stoneage here?)
Who can help or give hints? YOUR opinion is very appreciated!

please mail and post your reply.

Wolfgang Schechinger
Institute for Diabetes Research
Kölner Platz 1
80804 Muenchen
Germany
Phone +49 (89) 30 79 31 24
Fax            30 81 73 3
E-mail: DIABETES at LRZ.UNI-MUENCHEN.DE




More information about the Methods mailing list