DNA Reaction - Sequencing Single Stranded Templates


Jeff Lawrence

1. Aliquot DNA of the appropriate amount :
	Template		Amount
	========		=======
	Asymmetric PCR Product	200 ng
	Purified ss PCR Product	100 ng
	M13 DNA	2 µg
	Phagemid-induced ssDNA	2 µg
2. When ready to sequence, combine the Annealing Mix:
	6 µL	DNA, amount as above
	2 µL	5X Sequenase Buffer
	2 µL	Primer, about 1 pmol, or 2-10 ng.
3. Pulse down in a microcentrifuge Tube.

4. Incubate at 70° for 5 min to completely unwind DNA strands

5. Slowly cool to 37° over 20 min to anneal primer.

6. Proceed with Sequenase DNA Sequencing Protocol.
Last Update: Thursday June 19 2014
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Eric Kofoid eckofoid at ucdavis.edu