<aside>
📂
Unlisted protocol. This exists in the repo but was never added to the protocols index, so it has not been visible on the lab docs site.
</aside>
Partial restriction digest.
- Add the following to a 1.5 mL tube:
| Reagent |
Amount |
| Plasmid DNA |
1 μg |
| 10X Buffer |
2 μL |
| Enzyme 1 |
1 μL |
| Water |
To 20 μL |
- Incubate at the recommended temperature for 1 hour, or to completion.
- To the completed digest, add:
| Reagent |
Amount |
| 10X Buffer |
8 μL |
| Water |
72 μL |
- Label the tube "A" and place on ice.
- Aliquot 20 μL from A to tubes labeled "B", "C" and "D", and 10 μL to a tube labeled "E". "A" should have 30 μL remaining. Keep all tubes on ice.
- Add 1 μL of Enzyme 2 to "A". Mix well.
- Transfer 10 μL from "A" to "B". Mix well.
- Transfer 10 μL from "B" to "C", then 10 μL from "C" to "D", and 10 μL from "D" to "E". Mix well each time, and change tips between each transfer.
- Incubate all tubes at the recommended temperature for 30 seconds to 4 minutes. First try a 2 minute incubation. Place on ice.
- Add the first buffer from the PCR purification kit to each tube, then pool all the tubes. Perform the rest of the PCR purification.