minipreps for sequencing

Karen L. Yoas kyoas at bcm.tmc.edu
Sat Mar 5 13:25:35 EST 1994


In article <940304164627.35163 at UABCVSR.cvsr.uab.edu>, txpljfg at UABCVSR.CVSR.UAB.EDU writes:
> 
> Our largest problem so far has been stops at some of the GC rich areas.
>  The usual remedies (formamide gels, deaza analogs) haven't been as
> effective as we had hoped.  We intend using TdT to try and remedy this
> problem.  Any other ideas would be greatly appreciated.

I have been sequencing much larger areas than you are interested in but I
have had problems as well with GC rich areas.  Sometimes raising the rxn
temp to 42C works.  Other areas when I sequenced the opp strand, it came
out fine.  Weird.  For the rest I tried other methods for GC compressions
but with no luck until I tried the SequiTherm Cycle Sequencing Kit by
Epicentre Technologies.  Unbelievable.  Seqn I thought was impossible came
out beautifully.  They have sample kits which is not really expensive.  I
believe it was around $35.  I would have to look back on the form to make
sure.  The DNA I use when sequencing is CsCl purified so I don't know how
it would work with mini prep DNA.  The paperwork says it can obtain seqn
from "single bacterial colonies ... plagues ..." and various other "sources"
but it doesn't go into anymore detail than that.  Their 800 number is
#1-800-284-8474 if you are interested.  Good Luck!

Karen L. Yoas
Baylor College of Medicine
Department of Microbiology and Immunology





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