Smolke:Protocols/Digests: Difference between revisions
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*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 | *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. | **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 | *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 |
Revision as of 13:48, 15 October 2009
Josh's double digest mix
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). CIP
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