DNA Reaction - Digestion with 4 bp Site Restriction Endonucleases

Jeff Lawrence

1. Prepare DNA to exceptional purity. If necessary, include RNase and Proteinase K digestions, and additional phenol and chloroform extractions.

2. The following amounts of DNA are adequate for visualization:

	Source			Amount
	Eukaryotic chromosome	10 µg
	Bacterial chromosome	2 µg
	Lambda DNA		250 ng
	Plasmid DNA		100 ng
	PCR Product		100 ng

3. Digest the appropriate amount of DNA as follows:

	2 µL 	DNA
	2 µL	10X Reaction Buffer
	1 mL	2 mg/mL BSA
	1 mL  	Restriction endonuclease, @ 5 units/µg DNA
	14 mL	ddH₂O

4. Incubate at 37° for 6 hr to insure complete digestion.

5. Combine the following, in order:

20 µL Restriction digest 80 µL ddH₂O 2 µL 5 M NaCl 1 µL 1 % Linear Polyacrylamide 250 µL 100% Ethanol
6. Mix well and incubate at -20° for 2 hr. Centrifuge for 15 min at 4°.

7. Discard supernatant. Add 450 µL 70 % Ethanol. Rinse well, dislodging the pellet from the side of the tube. Centrifuge for 3 min at 4°.

8. Discard supernatant. Dry for 2 min in Speed Vac drier. DO NOT overdry.

9. Resuspend in 5 µL Sequenase Stop Solution dye. Load on polyacrylamide gel.

Last Update: Thursday June 19 2014

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Eric Kofoid eckofoid at ucdavis.edu