Diagnostic RP Digestion for Checking Insert Size

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This procedure is for checking the insert size of a BioBrick part. It uses digestion with EcoRI + PstI.

  • Label a 1.5 ml tube for each of the digestions to be set up
  • Transfer about 300 ng into each tube and make up the difference with pure dH2O to 6 ul. Or just use 6 ul of a miniprep
  • If you have fewer than 4 digestions, add the following to each tube:
  9 ul dH2O
  2 ul 2 NEB4 10X buffer
  2 ul 10X BSA
  0.5 ul HF EcoRI
  0.5 ul HF PstI
  • If you have 4 or more digestions, prepare a master mix as follows, where N is the number of digestions, then add 14 ul of it to each tube:
  9 x (N+1) ul dH2O
  2 x (N+1) ul 2 NEB4 10X buffer
  2 x (N+1) ul 10X BSA
  0.5 x (N+1) ul HF EcoRI
  0.5 x (N+1) ul HF PstI
  • Spin down contents of each tube
  • Incubate at 37 C for 10 minutes
  • Add 5 ul blue sample buffer to each and run on gel