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 
PCR products.

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
	protocol


*	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 

		Chris





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