DNA Reaction - Sequencing Double Stranded Templates - Nonidet Method


Jeff Lawrence

1. Purify DNA and resuspend in TE to a concentration of 100 ng/mL.

2. Prepare Denaturing Mix:
	1.0 µL	DNA (approximately 100 ng of a 1 kb PCR product; 1-5 mg plasmid)
	2.0 µL	1 µM Oligonucleotide (about 2 pmol)
	1.0 µL	5% Nonidet P-40 in water
	1.0 µL 	ddH2O
3. Incubate at 100° for 3 min. Immediately place on dry ice for 5 min.

4. Thaw during a 15 sec spin and transfer to ice

5. Continue with Sequenase sequencing, using this mixture as the Annealing Mix.
Last Update: Thursday June 19 2014
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Eric Kofoid eckofoid at ucdavis.edu