DNA Reaction - Digestion with 8 bp Site Restriction Endonucleases

Jeff Lawrence

1. Prepare DNA in Agarose Plugs.

2. Rinse plugs for 60 min at 22° twice with large volumes of 1 X Restriction Buffer without Mg++.

3. Discard buffer. Place plug in 1.5 mL microcentrifuge tube. Add 1 X Mg++-free Restriction Buffer to cover plug.

4. Add 5 µL Restriction endonuclease and incubate at 15° for 30 min with swirling.

5. Add MgCl2 to a final concentration of 2.5 mM.

6. Incubate at appropriate temperature overnight.

7. Remove buffer with a pipet tip.

8. Cleave plug into 2 portions. Load one portion into slot in agarose gel.

9. Cover with agarose. Electrophorese.

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