IGEM:PennState/2006/Restriction

Restrictions

Time: 20 min

Reference: NEB.com

Pre

• (optional) Determine plasmid concentration via A260 measurements or by comparing intensities of bands on gel with those of the ladder DNA (whose masses are known).

Restricting

1. To an eppendorf tube, add:
   1. Appropriate volume of plasmid for a total of approx. 700 ng DNA (usually, if plasmid prep is good, this will be 2 μL (i.e. ~350 ng/ μL)).
   2. 1 μL of appropriate 10X concentration buffer (to determine correct buffer check compatibility of restriction buffers using NEB catalogue or going online to NEB.com [1])
   3. If necessary (i.e. check NEB), add 1 μL 10X concentration BSA
      • BSA~bovine serum albumin
   4. [x] μL of dH2O to make the total reaction volume in tube of 10 μL.
   5. Chosen restriction enzymes. They are at a high enough concentration that 0.5 μL of each is more than sufficient for a restriction digest. ALWAYS ADD THESE LAST (and work quickly), in order to minimize time out of the freezer. Keep these enzymes in their low-temp blue carrying case when out of the freezer.
2. (Gingerly) flick tubes, and spin down in microcentrifuge for a second
3. Incubate at 37°C (in water bath or warm room) for 1-4 hrs. (1-2 hrs. optimal)
4. (Under review)...treat any vectors w/0.2 μL CIP and incubate for 30 min at 37°C with rest of restrictions

Suggested NEBuffers for Double Digestion

http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp

Cloning

Main