MIT iGEM Restriction Digests

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(Materials)
Current revision (13:05, 24 June 2008) (view source)
(Method)
 
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#MIX THE REACTION BY PIPETTING HALF THE VOLUME UP AND DOWN.
#MIX THE REACTION BY PIPETTING HALF THE VOLUME UP AND DOWN.
#Incubate the reaction at 37*C for 2hrs to ensure complete digestion.  
#Incubate the reaction at 37*C for 2hrs to ensure complete digestion.  
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#Deactivate the enzymes by heating for 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.
+
#Deactivate the enzymes by heating @ 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.
-
#Keep digest at -20*C or run immediately on gel.
+
#Store digest at -20*C or run immediately on gel.

Current revision

Restriction Digest protocol

For Analytical digests, aim for 500-1000ng of DNA; for a preparative digest (where you know you want to cut out a band) try to load as much DNA as possible (gel lanes w/ wide-toothed combs can hold up to 50µl).

Materials

  • DNA; the thing you want to cut. Usually plasmid or PCR product. Measure concentration in Nanodrop beforehand.
  • Appropriate NEB 10x Buffer (check the NEB enzyme chart or catalogue to find compatible buffers).
  • Appropriate enzymes.
  • ddH2O
  • BSA (100x from NEB)

Method

The following volumes apply to a 20µl analytical digest; for larger, preparative digests, simply scale up (eg. for a 30µl digest, use 3µl of 10x buffer, etc)

  1. Add 20µlTotalVolume-(µlDNA + µlBuffer +µlBSA +µlEnzyme)µl ddH2O to PCR tube. (eg. 20µl-(1µlDNA+2µlBuffer+0.2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=15.8µl ddH2O)
  2. Add 0.2µl BSA to tube.
  3. Add 2.0µl 10x Buffer to tube.
  4. Add appropriate amount of DNA to tube.
  5. Add 0.5µl of each enzyme to tube.
  6. MIX THE REACTION BY PIPETTING HALF THE VOLUME UP AND DOWN.
  7. Incubate the reaction at 37*C for 2hrs to ensure complete digestion.
  8. Deactivate the enzymes by heating @ 80*C for 20min (in the thermocycler). This will inactivate most enzymes, though check to make sure that your enzyme can be heat deactivated by checking in the NEB catalogue.
  9. Store digest at -20*C or run immediately on gel.
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