USTC/Notebook/Restriction Digests

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Restriction Digests

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)

Procedure

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)

add the following components to PCR tube:

  • 2µl BSA (diluted to 10X beforehand)
  • 2µl 10x Buffer
  • appropriate amount of DNA
  • 0.5µl of each enzyme
  • appropriate amount of ddH2O to PCR tube. (20µlTotalVolume-(BSA + Buffer + DNA + Enzyme)µl

eg. 20µl-(5µlDNA+2µlBuffer+2µlBSA+0.5µlEnzymeA+0.5µlEnzymeB)=10µl ddH2O)

  • MIX the reaction by stiring
  • Incubate the reaction at 37*C for 2-4hrs to ensure complete digestion.
  • Store digest at -20*C or run immediately on gel.
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