DNA Reaction - Sequencing Double Stranded Templates - Alkaline Denaturation


Jeff Lawrence

1. Purify DNA and resuspend in TE to a concentration of 10 ng/ mL.

2. Prepare Denaturing Mix:
	10.0 µL	DNA (approximately 100 ng of a 1 kb PCR product; 1-5 mg plasmid)
	4.5 µL	2 N NaOH
	26.0 µL	ddH2O
3. Mix by vortexing and incubate at 22° for 3 min.

4. Combine:
	40.5 µL	Denaturing Mix
	1.5 µL	1% linear polyacrylamide carrier
	4.5 µL	4 M Ammonium Acetate, pH 4.6
	120.0 µL	100% Ethanol
5. Mix well prior to adding ethanol. Mix and incubate at -20° for 30 min.

6. Centrifuge for 15 min at 4°. Discard supernatant. Add 450 µL 70% ethanol.

7. Centrifuge for 2 min at 4°. Discard supernatant.

8. Dry for 2 min in Speed-Vac. DO NOT OVERDRY.

9. When ready to sequence, combine the Annealing Mix:
	1	Pellet of 100 ng denatured DNA
	6 µL	ddH2O
	2 µL	5X Sequenase Buffer
	2 µL	Primer, about 1 pmol, or 2-10 ng.
10. Mix well to resuspend. Incubate at 37° for 10 min to anneal primer.

11. Proceed with Sequenase DNA Sequencing Protocol.

Last Update: Thursday June 19 2014
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Eric Kofoid eckofoid at ucdavis.edu