Richard Lab:Restriction Digest
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If you are needing to know more
If you are needing to know more the [[Restriction_Digest | other restriction digest protocols]].
Revision as of 15:16, 12 June 2009
This protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration:
- Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning
- Add the following in a micro-centrifuge tube
- 5μl of Buffer 2
- 1μl of BSA
- 42μl of DNA solution (Dilute PCR products in half)
- Vortex Enzymes and add 20 units (1μl) of each to the tube
- Incubate reaction in a 37°C water bath for at least one hour (I like 1:45).
- Heat kill the digest for 15 minutes at 75°C.
- If digesting a vector add 10 units (1µl) phosphatase) and 5µl Phosphatase Buffer and incubate an additional 45 minutes at then heat kill againat 75°C for 15 min.
- Store digested DNA in the freezer (-20°C).
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- If you're not getting good digestion it might be because your enzyme is bad. Double the digestion time and see.
- Longer digest gives more complete digestion, especially if you have >1µg of DNA, but can sometimes give nonspecific digestion
If you are needing to know more then you can check out the other restriction digest protocols.
or instead, discuss this protocol.