Smolke:Protocols/Digests

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Josh's double digest mix

  • 1.5ug vector
  • 2uL 10x buffer
  • 0.2uL 100x BSA
  • 0.5uL each enzyme
  • Water to 20uL

Run overnight at 37C.

Adjust this mix depending on whether you're making a master mix. For one reaction, I up the BSA to 0.5uL (since I don't trust pipetting 0.2uL). For a master mix, you can judiciously reduce the amount of enzyme (<0.5uL/rxn) and adjust the relative enzyme amounts depending on the enzyme concentration (so use more an an enzyme at 5 U/uL and less of one at 20 U/uL).

Note: You can digest with some enzymes in as little as 1 hour, just make sure to use 1-2 ug of DNA max to help ensure that DNA is double cut.-SG

CIP

  • CIP treat most vectors after digestion. First heat inactivate the enzymes (restriction enzymes bind to the cut ends of the DNA and compete with CIP), then add CIP. Incubate 1 hour at 37C
    • Note that CIP doesn't work well in Buffer 1. If you absolutely need to do your digest in that buffer and then CIP treat, you should clean up the digest and switch to a different buffer.
    • CIP treating is mostly useful in preventing single-cut vectors from re-ligating to itself. If you are performing a double-cut digestion with a significantly sized cutout, such that you could gel extract a vector you are certain is double cut, then it is not necessary to treat it with CIP. --YC
  • NEB recommends 0.5 U CIP/ug vector. Stock concentration is 10 U/uL. So, for a 1.5ug digest, you need ~1 U or 0.1uL. That's hard to pipet. For multiple digests, I'd make a master mix of dilute CIP, then add 1uL of that to each digest. For a single digest, I use as little as I'm comfortable pipetting, or ~0.5uL. -Josh
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