Help:Protocols/Restriction Digest

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Overview

When using parts for 3A Assembly, or testing the quality of a part you'll need to run a restriction digest. We recommend using the following protocols.

Restriction Digest Protocol

estimated time: 30 min. active, 50 min. incubation

The following protocol assumes you are simply doing a restriction digest for quality control, you can use the protocol below.

If you'll be doing restriction digests for 3A assembly, see the 3A assembly protocol or linearized plasmid backbone protocol.

Restriction Digest from iGEM Videos.


Materials

  • (1) 8-tube strip, or (3) 0.6ml thin-walled tubes
  • BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul)
  • dH2O
  • NEB Buffer 2
  • BSA
  • Restriction Enzymes: EcoRI, SpeI, XbaI, PstI


Notes:

  • You should keep all materials on ice.
  • At iGEM HQ we use restriction enzymes from New England Biolabs

Equipment

  • Ice and bucket/container
  • Thermal cycler or heating block

Method

  1. Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.
  2. Add 2.5ul of NEBuffer 2.
  3. Add 0.5ul of BSA.
  4. Add 0.5ul of EcoRI.
  5. Add 0.5ul of PstI.
  6. There should be a total volume of 20ul. Mix well and spin down briefly.
  7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. We incubate in a thermal cycler with a heated lid
  8. Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.