Direct sequencing of PCR products
Christopher Paul Palmer
sasplmrc at reading.ac.uk
Wed Mar 1 10:36:06 EST 1995
I have obtained very reliable results from direct sequencing of
1. Purify PCR product from 100-200 ul of reaction using
Geneclean, and elute into 30 ul of TE.
2. Mix for each template 5 ul PCR product
2 ul Primer
2 ul Sequenase 5 x reaction buffer
1 ul DMSO
3. Boil for 5 mins, and then snap cool on ice.
4. Follow the protocol as described for Sequenase sequencing of
plasmids after the denaturation and annealing of plasmid/primer
* The only difference is that the termination mixtures are made up
containing 10% DMSO.
* Also the termination reactions are performed at 50 C.
* The primer concentration are those recommended by the Sequenase
* If you dont use Geneclean then you could substitute any other PCR
clean up procedure, e.g Promega PCR purification resin/columns.
Best of luck
More information about the Methods