Difference between revisions of "Help:Protocols/Restriction Digest"
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When using parts for 3A assembly, or testing the quality of a part you'll need to run a restriction digest. | When using parts for 3A assembly, or testing the quality of a part you'll need to run a restriction digest. | ||
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Revision as of 19:03, 17 May 2012
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Restriction Digests |
| When using parts for 3A assembly, or testing the quality of a part you'll need to run a restriction digest. |
Contents |
At iGEM HQ we recommend using the following protocols. The large reaction protocol will have enough of a volume for ligation and you'll be able to use a portion to run a gel. The small reaction protocol will only have enough volume for
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.
We incubate in a thermocycler with a heated lid
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.
Linearized Plasmid Backbones Original Protocol