MIT iGEM Restriction Digests: Difference between revisions
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Felix Moser (talk | contribs) (New page: =Restriction Digest protocol= ==Materials== *DNA; the thing you want to cut. *Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers). *Appropriate...) |
Felix Moser (talk | contribs) |
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*Appropriate enzymes. | *Appropriate enzymes. | ||
*ddH2O | *ddH2O | ||
*BSA ( | *BSA (100x from NEB) | ||
==Method== | ==Method== |
Revision as of 09:47, 24 June 2008
Restriction Digest protocol
Materials
- DNA; the thing you want to cut.
- Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers).
- Appropriate enzymes.
- ddH2O
- BSA (100x from NEB)
Method
The following is for a 20µl analytical digest; for larger, preparative digests, simply scale up
- Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube.
- Add 0.2µl BSA to tube.
- Add 2.0µl 10x Buffer to tube.
- Add appropriate amount of DNA to tube.
- Add 0.5µl of each enzyme to tube.
- MIX THE REACTION BY PIPETTING HALF THE VOLUME UP AND DOWN.
- Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
- Deactivate the enzymes by heating for 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.
- Keep digest at -20*C or run immediately on gel.