HELP!sequencing

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Tue Dec 27 11:28:22 EST 1994


Elena Baena Gonzalez writes:

> I'm having serious problems with sequencing. I've been using the 
> Circumvent thermal cycle dideoxyDNA sequencing kit (New England 
> Biolabs) for sequencing PCR products(dsDNA)of the heterozygous 
> plant Cardaminopsis petraea. I get some sequence, but with so much 
> background and compression that it is impossible to read it.
> ...[could it be heterogeneous template?]

Look and see if there is vertical distortion in the band spacing.
If so, this has to do with secondary structure in the seq. related
to G+C content.  Use procedures designed to seq. G+C rich DNA.

If not, maybe the template is heterogeneous.  The quickest way to
know is to clone some and seq. a few clones.

Hope this helps.
Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu




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