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Restrictionsdigest
(Source: http://www.promega.com/enotes/applications/ap0078.htm, http://openwetware.org/wiki/Engineering_BioBrick_vectors_from_BioBrick_parts/Restriction_digest)
Restriction enzyme digestions were assembled as follows:
plasmid DNA 1µg
restriction enzyme 1µl
10X buffer 2µl
BSA (10µg/µl) 0.2µl
nuclease-free water to final volume 20µl
Table 1. Restrictionenzymes with data about their activity in selected buffers. Optimal also taken from following webpage especially if double digestion wanted: http://www.promega.com/guides/re_guide/research.asp?search=buffer
| Enzyme | Activity in buffer A | Activity in buffer B | Activity in buffer C | Activity in buffer D | Activity in buffer H | Activity in buffer MC |
|---|
| XbaI | 50-75 | 75-100 | 75-100 | 100 | - | 100 |
| EcoRI | 25-50 | 50-75 | 50-75 | 50-75 | 100 | 100 |
| PstI | 10-25 | 50-75 | 50-75 | 50-75 | 100 | 25-50 |
| SpeI | 75-100 | 100 | 75-100 | 75-100 | - | 100 |
Procedure
Vortex all reagents before use. (especially the enzymes)
1. Add appropriate amount of deionized H2O to sterile 0.6 mL tube
2. Add restriction enzyme buffer.
3. Add BSA.
4. Add DNA.
5. Add each enzyme.
Also, the enzyme is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 1 μL, just touch your tip to the surface of the liquid when pipetting.
6. Incubate for 2 hours at 37°C.
7. Incubate for 20 mins at 80°C to heat inactivate enzyme.
This step is sufficient to inactivate even Pst I.
8. Incubate 4°C until you pull the reaction out of the thermal cycler. (Thermal cycler can be used for a installed program for digestion, even overnight)
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