DNA Preparation - Single Stranded DNA by Exonuclease Digestion

Jeff Lawrence

1. Prior to the PCR, kinase one oligonucleotide, corresponding to the strand to be digested, with nonradioactive ATP.

2. Execute the PCR. Extract and precipitate the product to remove TAQ polymerase.

3. Resuspend the product in 100 µl Exonuclease Buffer:

	67 mM	Glycine, pH 9.4	(67 mL	1 M Glycine, pH 9.4)
	3 mM	MgCl₂		(3 mL	1 M MgCl₂)
				(930 mL	ddH₂O)

4. Add 4 units Lambda Exonuclease. Incubate at 37° for 15 min.

5. Extract and precipitate the single stranded DNA.

6. Resuspend the DNA in an appropriate amount of TE for DNA sequencing.

7. The kinased oligonucleotide may be used for DNA sequencing.

Last Update: Thursday June 19 2014

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Eric Kofoid eckofoid at ucdavis.edu