Richard Lab:Restriction Digest

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Contents

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-----

Materials

  • Prepared DNA from miniprep, PCR, or Gel Extraction
  • Restriction Endonucleases
    • With corresponding 10X buffer. NEBuffer 2 can be used for most applications but check this chart to be sure.
  • BSA (optional)
  • Phosphatase
  • Distilled water

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. 1μl of BSA
    3. 40μl of DNA solution (Dilute PCR products 1:4)
  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
  • Beware the the NEB double digest chart. For EcoRI and PstI double digest it recommends using the EcoRI NEBuffer. However based on my digests both NEBuffer 2 and 3 work better; with NEBuffer 2 giving the most complete double digestion. It is funny that thye recommend the Eco buffer because they also say that Pst only exhibits 50% activity with that buffer.

References

For more information you can check out the other restriction digest protocols.

Contact

or instead, discuss this protocol.

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