Arkin:JCAProtocols Digesting

Protocols Page

Set up the following digestion reactions, one reaction for each plasmid. The choice of NEB Buffer (1 through 4) depends on the enzymes used and can be found at NEB http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp

For SpeI, XbaI, AlwNI, EcoRI, PstI, BsaI you would use NEB Buffer 2.

• 3 uL ddH2O
• 5 uL miniprep DNA
• 1 uL NEB Buffer
• 0.5 uL of Restriction Enzyme 1
• 0.5 uL of Restriction Enzyme 2

1. Incubate for 2 hrs at 37 degrees
2. load entire reaction on an agarose gel and run it
3. cut out the bands on the UV box
4. proceed to gel purification