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Protocol/5
From 2010.igem.org
Protocol 5: Restriction Digest
Reagents:
- Restriction enzyme buffer
- Buffer information can be found at www.NEB.com using the NEBtools.
- Restriction enzymes
- MilliQ water
- DNA to be digested
Procedure:
- Select a restriction enzyme buffer that is appropriate for BOTH of the enzymes you are using. See information sheets at front of lab for correct buffer and concentration.
- The total volume of all enzymes in the reaction should be less than 10% of the final reaction volume. Enzymes usually are supplied at 10U/ul and 1ul will be more than enough for our digests.
- Add components in the following order:
- Water
- Buffer
- Bovine Serum Albumin-BSA (if needed)
- DNA
- Enzyme I
- Enzyme II
- An example of a typical 25 ul reaction would be:
| MilliQ water | 15.5 ul |
| 10x NEBuffer | 2.5 ul |
| DNA (200 ng/ul) | 5.0 ul |
| XbaI | 1.0 ul |
| PstI | 1.0 ul |
- Incubate at 37oC for one hours (longer is okay too).
Notes:
FastDigest (by Fermentas) enzymes use a single uniform buffer, and claim to work in 5 minutes.
