IUBio

Plasmid problems and Big-Dye

Shaun Tyler styler at hpb.hwc.ca
Tue May 19 18:09:58 EST 1998


We have recently switched over to the Big-Dye chemistry and I have to
admit that I=92m pretty impressed.  However I=92ve recently come across
something which leads me to believe that the new chemistry may not be
quite as robust as the old one.   I=92ve been having some problems with
sequencing some PCR products cloned into pCRII.  The amplicons are
approximately 700 - 800 bp and the plasmids are being purified using
Promega=92s Wizard Plus system.  I=92ve sequenced a number of similar clo=
nes
in the past with the old chemistry using exactly the same procedure and
the results were great.  But now I=92m getting either very weak signal or
no signal at all.  The plasmid preps are quantitated by A260 and I=92m
using the same amount as before.  I=92ve had some moderate success in
improving results by using the cycling conditions for cosmids (i.e.
increasing the cycles to 60) recommended by  Bruce Roe (I=92ve been using
this for sequencing some rather large plasmids and stumbled on this by
accident -- you can probably guess how).  I=92ve also had some luck by
simply reprecipitating the plasmids with EtOH after extraction.  I
suspect that there may be something in the purified plasmid obtained
with the Wizard kit which is partially inhibiting the sequence reaction
but this was never a problem with the old FS chemistry.

Any comments or suggestions you may have would be appreciated.



Shaun Tyler
DNA Core Facility
Laboratory Centre for Disease Control
Health Canada

Ph#:  (613) 941-6441
FAX#: (613) 957-1358

E-mail:  styler at hpb.hwc.ca






More information about the Autoseq mailing list

Send comments to us at biosci-help [At] net.bio.net