Lissa1:Plasmid Digests

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.