User:Eric Ma/Notebook/iGEM 2010 Lab Prep/2010/05/13

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Restriction Enzyme Test

  • To test restriction enzymes, digest DNA with E+P, X+S, E+S, X+P; in addition to this, do four single cuts.
  • We can tell which enzyme is not working (if it is such) by examining which pairs are not cutting properly.
  • I will cut Jason's miniprepped DNA because it had the most amount of DNA present.
  • 4 double digests + 4 single cuts, for further confirmation.
  • Volumes used:
Reagent Control E X S P ES EP XS XP Master Mix
10X BSA 2 2 2 2 2 2 2 2 2 20
10X Buffer 2 2 2 2 2 2 2 2 2 2 20
EcoRI 0 1 0 0 0 1 1 0 0 N/A
XbaI 0 0 1 0 0 0 0 1 1 N/A
SpeI 0 0 0 1 0 1 0 1 0 N/A
PstI 0 0 0 0 1 0 1 0 1 N/A
DNA 3 3 3 3 3 3 3 3 3 30
sdW 30 30 30 30 30 30 30 30 30 300
Total 37 38 38 38 38 39 39 39 39 370
  • Digest for ~1-2 hrs (not so crucial) at room temp.
  • Note: Initially wanted only 10 µl of water, but in the end, I calculated for 30 µl instead in the master mix for a total of 300 µl of water. Therefore, the reaction may be a bit dilute, which is why I will let this run while I continue writing and rehearsing the podcast instead. The timing shouldn't be too important.
  • Digest ran overnight on ice after ~2h incubation in 37ºC.