Difference between revisions of "Help:Protocols/Restriction Digest"
(New page: 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> 3. Add 0.5ul of BSA to the tube.<br> 4. Add 1ul of you...) |
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| + | At iGEM HQ we use this protocol for restriction digests, which will be used further down the line for running on gels and ligations. | ||
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> | ||
Revision as of 23:11, 27 September 2010
At iGEM HQ we use this protocol for restriction digests, which will be used further down the line for running on gels and ligations.
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.