DNA Reaction - Digestion with Typical Restriction Endonucleases

Jeff Lawrence

1. Prepare DNA and resuspend in an appropriate volume of TE.

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

	Source			Amount
	Eukaryotic chromosome	2 mg
	Bacterial chromosome	200 ng
	Lambda DNA		50 ng
	Plasmid DNA		20 ng
	PCR Product		20 ng

3. Choose the appropriate reaction buffer for each restriction enzyme.

4. Combine the following:

	1 µL	DNA
	2 µL	10X Reaction Buffer
	1 µL 	Restriction enzyme, @ 3 units/µg DNA
	16 µl	ddH₂O

5. Incubate at the appropriate temperature for 2 hr. If temperature is 50° or greater, cover the reaction mixture with 20 µL Light Mineral Oil.

6. To electrophorese, add 4 µL Stop Dye to the side of each tube:

	30 %	Glycerol		6 mL	50 % Glycerol
	5 mM	EDTA			100 mL	500 mM EDTA, pH 8.0
	0.07 %	Bromophenol Blue	7 mL	Bromophenol Blue
	0.07 %	Xylene Cyanol		7 mL	Xylene Cyanol
					3.9 mL	ddH₂O

7. Centrifuge for 5 sec to pool. Load on gel.

Last Update: Thursday, 12-Dec-2024 00:55:25 UTC

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Eric Kofoid eckofoid at ucdavis.edu