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.
 * 5) 	Add 3 uL of the buffer (it’s at 10x concentration).
 * 6) 	Add BSA (.3 uL, you may need to make a 10x solution and add 3 uL instead)
 * 7) 	Add 1 uL of enzyme.
 * 8) 	Add DNA. We used 2 ug of DNA.
 * 9) 	Add water to final volume of 30 uL.
 * 10) 	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.
 * 11) 	Run on small agarose gel with uncut plasmid as a control to check for cutting.
 * 12) 	Prepare samples to go in gel.
 * 13) 	You need blue buffer 1x (2 blues, sorbitol) which is in 4 degree freezer in little tubes (brown tubes, because of light sensitivity).
 * 14) 	Put equal amounts buffer and DNA on parafilm to load in to gel. Use 4 uL each.  (You want 200 ng, approximately).
 * 15) 	Also use DNA ladder.