ds sequencing troubles

jo_c at vcp1.vcp.monash.edu.au jo_c at vcp1.vcp.monash.edu.au
Thu Feb 17 21:39:53 EST 1994


I have been having trouble with ds sequencing of almost all
my plasmids but especially a pGEX construct.  My insert (849BP)
is 68% GC rich and I'm wondering if this is causing problems.
My sequencing gels either give me nothing or bands in all lanes.
I've tried using a control without my insert and I get faint
bands but they seem to be in all the right places.  I have 
tried using both the Sequenase kit and Taq track by Promega
and have varied the primer concentrations from 2pmol upto 
about 10pmol.  The GST primer I've been using is a home made
one which hasn't been purified.  Is this a problem?  I've 
used other unpurified home made primers on ss sequencing and
they have worked fine.  I have used standard Maniatus alk. lysis,
acid phenol and I'm currently using home made mini-prep resin
(its works as good as the Magics/Wizards) as sources for the 
DNA.  Can anyone help, or should I just go back to ss sequencing?

Thanks in advance

Jo Caine                              
Victorian College of Pharmacy
Melboune, Australia
EMAIL:  JO_C at VCP.MONASH.EDU.AU




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