PCR-sequencing

Jeffrey Vance jmv at galactose.mc.duke.edu
Thu Mar 31 22:55:07 EST 1994


schoenbaechl at urz.unibas.ch wrote:

: I am planning to sequence a 1 kb piece from human DNA. I want to use a ds-DNA
: PCR sequencing kit from Gibco. Do I first have to amplify this fragment or can
: I PCR-sequence directly from DNA isolated from blood ?
[couple of lines deleted]

No, yes.
If the kit contains phosphatases & nucleases for prior cleaning up of the PCR
itself, you can do your reaction & remove some directly for sequencing. 
If not, you can excise the product from an agarose gel, Sephaglas or 
agarase it etc., & use the recommended volume if the concentration is OK.
USB recommends 50-500ng for PCR products in <10ul; for 1kB, it could be 
100-500ng. I find the rx differ according to primer; some anneal better 
within different windows of concentration of template. 

I have sequenced products at & over 1kB from human genomic DNA using the 
same primers as for PCR & 5ul of a 25ul rx from which 15ul run out on an 
agarose gel yields a bright band.

Bon chance!

*DB*
***DUMC Neurology***





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