Help:Protocols/Restriction Digest

Revision as of 14:02, 28 September 2010 by Vinoo (Talk | contribs)

At iGEM HQ we use this protocol for restriction digests along with enzymes purchased from NEB.

Materials

  • PCR tube
  • dH20
  • Enzymes (EcoRI, XbaI, SpeI, PstI)
  • BSA
  • Enzyme Buffer (NEBuffer 2)*

Notes: You should keep all materials on ice.

Protocol

1. Add 500ng of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.
2. Add 5ul of NEBuffer 2 to the tube.
3. Add 0.5ul of BSA to the tube.
4. Add 1ul of your first enzyme.
5. Add 1ul of your second enzyme.
6. There should be a total volume of 50ul. Mix well and spin down.
7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.
8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2ul of the digest (20ng of DNA) for ligations.