Digestions
Reaction Mix:
- 500 ng – Template
- 5 µl – 10x Buffer
- 1 µl – NEB Enzyme 1 (10 Units/µl) = 10 Units in 50 µl reaction
- 1 µl – NEB Enzyme 2 (10 Units/µl) = 10 Units in 50 µl reaction
- dH2O to 50 µl
- TOTAL – 50 µl
Procedure:
1. Calculate how much template is needed for digestion. Then calculate how much water needs to be added to make a 50 µl reaction.
2. Pipette appropriate amounts of dH20, template, and 10x Buffer in that order into a PCR tube (200 µl). Mix reagents together by vortexing and then tapping tube on desk to keep reagents on the bottom of the PCR tube.
3. Add appropriate amounts of Enzyme 1 and Enzyme 2. Once added gently swirl around reaction mix with pipette tip.
4. Incubate at 37o C for 1 – 2 hours
5. Incubate at 80 oC for 20 minutes to deactivate the restriction enzymes
6. Run a gel of your digestions on a gel with low temperature melting agarose. Run at 100V for 1 hr. 30 min.
7. Look at gel under 320nm UV light. Conduct gel extraction purification.
NOTE: When digesting your vector it may be useful to use 2 µl of each enzyme in the 50 µl reaction and extending incubation time.
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