Lissa1:Plasmid Digests

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  1. Find prepped plasmid (you purified it out of E. coli using a QIAGEN kit – see protocol for doing that).
  2. Get restriction enzymes. They are in the -20 freezer.
  3. Get buffers from -20 freezer. You find out which buffers you need form the NEB catalog. You might also need BSA.
  4. Make 30 uL of total digest.
    1. Add 3 uL of the buffer (it’s at 10x concentration).
    2. Add BSA (.3 uL, you may need to make a 10x solution and add 3 uL instead)
    3. Add 1 uL of enzyme.
    4. Add DNA. We used 2 ug of DNA.
    5. Add water to final volume of 30 uL.
  5. Put in thermal cycler. 2 hours for cutting to happen, then look at NEB catalog to find out how long/at what temp to heat for inactivation.
  6. Run on small agarose gel with uncut plasmid as a control to check for cutting.
    1. Prepare samples to go in gel.
      1. You need blue buffer 1x (2 blues, sorbitol) which is in 4 degree freezer in little tubes (brown tubes, because of light sensitivity).
      2. Put equal amounts buffer and DNA on parafilm to load in to gel. Use 4 uL each. (You want 200 ng, approximately).
      3. Also use DNA ladder.