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