Richard Lab:Restriction Digest

Overview

This protocol is typically used to do bio-brick digests with the restriction sites consisting of the following configuration:

-----EcoRI--XbaI--Part--SpeI--PstI-----

Procedure

1. Quickly vortex all ingredients (Buffer, BSA, DNA, Enzymes) before beginning
2. Add the following in a micro-centrifuge tube
   1. 5μl of Buffer 2
   2. 1μl of BSA
   3. 42μl of DNA solution (Dilute PCR products in half)
3. Vortex Enzymes and add 20 units (1μl) of each to the tube
4. Incubate reaction in a 37°C water bath for at least one hour (I like 1:45).
5. Heat kill the digest for 15 minutes at 75°C.
6. 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.
7. 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

• user:Michael A. Speer

or instead, discuss this protocol.