sequenasePCR

Alistair Forrest forrest at biosci.uq.oz.au
Fri Mar 18 23:53:54 EST 1994


ckrudy at bimcore.emory.edu (Margaret M Lanterman) writes:


>I am trying to sequence a PNCR product of about 200bp, is this too small for sequenase direct sequencing?
>help!
>Margaret Lanterman

This shouldn't be too small for sequencing, the problem probably lies in the
difficulty of sequencing PCR products directly (in general). If you must use
sequenase then you have to make well and truly sure your dna is denatured and
stays denatured. NaOH and sometimes heat treatment will help (best dig up some
references). To get in close you can use the Mn buffer supplied in the sequenase
kit.
	Personally I would avoid sequenase for sequencing a pcr product. I have
used ABIs Dye-Terminator sequencing (Taq cycle sequencing) to routinely sequence
short PCR products with much greater success than with sequenase. If you must
use manual sequencing then you could try the fmol cycle sequencing kit. (I thinkit's put out by promega). Cycle sequencing will ensure you keep the template
available for the primer to bind, and it overcomes secondary structure.

	Hope this helps,
			Catch ya later,
					Alistair Forrest







 



More information about the Methods mailing list