DNA Reaction - Digestion with Typical Restriction Endonucleases
1. Prepare DNA and resuspend in an appropriate volume of TE.
2. The following amounts of DNA are adequate for visualization:
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, 19-Jun-2014 11:50:24 PDT
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