|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.
- Ice and bucket/container
- (1) 8-tube strip, or (3) 0.6ml thin-walled tubes
- BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul)
- NEB Buffer 2
- Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
- Thermal cycler
- You should keep all materials on ice.
- At iGEM HQ we use restriction enzymes from New England Biolabs
- Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.
- Add 2.5ul of NEBuffer 2.
- Add 0.5ul of BSA.
- Add 0.5ul of EcoRI.
- Add 0.5ul of PstI.
- There should be a total volume of 20ul. Mix well and spin down briefly.
- 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
- Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.