Difference between revisions of "Help:Protocols/Restriction Digest"
| Line 1: | Line 1: | ||
| − | At iGEM HQ we use this protocol for restriction digests | + | 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.<br> | 1. Add 500ng of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.<br> | ||
2. Add 5ul of NEBuffer 2 to the tube.<br> | 2. Add 5ul of NEBuffer 2 to the tube.<br> | ||
| Line 8: | Line 18: | ||
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> | ||
| − | 8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. | + | 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 14:02, 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.
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.