sequencing pcr products

Chris LaRosa clarosa at biocomp.unl.edu
Fri Apr 7 09:26:30 EST 2000


> We are going to use pcr product as our template to sequence.  Is too
> much primer in the sequencing reaction a bad thing?
> 

We routinely sequence pcr products.   It is necessary to get rid of the
nonDNA reagents and products, and we find that the Gibco Concert PCR
cleanup kits work well and cheaper than Qiagen .  For the ABI BigDye
prism chemistries.  It is our experience that Absorbance at A260 is not
sensitive enough.  We now use a fluorimeter 
with the Hoescht ___ (sic ) dye.   You can also quantitate the pcr
product by running on gels and estimating by eyeball, or spotting ETBR
stained dna  onto saran wrap and quantitating against a standard
series.  
Quantitation of the DNA in ABI chemistries is critical perhaps unlike
many  other sequencing protocols which are more tolerant of wide
template concentrations.




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