ABI Plasmid sequencing

Stock up and save. Limit one. pkmartin at netcom.com
Tue Apr 11 18:19:25 EST 1995


Michael Gorry (mcgorry at med.pitt.edu) wrote:
: I have recently tried to sequence two plasmids with complete failure.

(intervening stuff removed)

mike... (you gave up at two?)

assuming that your are using all ABI's kits and conditions (i.e. the
PRISM DyeDeoxy Terminators) and a PEC 9600, I can tell you that I've
had lousy results following the protocols exactly as published in 
the instruction manual (the pGEM3Zf+ ds control gave no signal when
sequenced with their machine and their protocol exactly as written).

i can tell you that once i altered the thermocycling conditions (i.e.
a longer intial denaturation step, presumably to denature plasmid
DNA more effectively), that i got satisfactory results.  i too 
compared plasmid vs. PCR-fragment sequencing and found that 
optimal thermocycling conditions for DNA sequencing differ between the two
and seem to correspond loosely to the total length of your template
( a 5 kB plasmid will have different annealing kinetics than a 
400 bp PCR product ). 

keep in mind that the exact cycling parameters will depend on your
thermocycler.  ALSO: even though you will sacrifice flexibility in
your choice of primers, i would highly recommend that you move to the
dye primer chemistry which will give you much more reliability in 
data acquisition.

contact me directly if you need more assistance.
.doc





More information about the Methods mailing list