Difference between revisions of "Help:Protocols/Restriction Digest"
| Line 17: | Line 17: | ||
5. Add 1ul of your second enzyme.<br> | 5. Add 1ul of your second enzyme.<br> | ||
6. There should be a total volume of 50ul. Mix well and spin down.<br> | 6. There should be a total volume of 50ul. Mix well and spin down.<br> | ||
| − | 7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.<br> | + | 7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.<br> ''We incubate in a thermocycler with a heated lid''<br> |
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. | 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. | ||
Revision as of 14:03, 28 September 2010
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.
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.