Richard Lab:Restriction Digest: Difference between revisions
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or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | ||
[[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]] | [[Category:Protocol]] [[Category:DNA]] [[Category:In vitro]] |
Revision as of 12:15, 12 June 2009
Overview
This protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration:
Procedure
- 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).
Notes
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
References
If you are needing to know more see the other restriction digest protocols.
Contact
or instead, discuss this protocol.