Goal:

To cut dsDNA using restriction enzymes for analysis (analytical scale digest).
We use both NEB and Fermentas enzymes, so protocols for both are below.

Protocol 1: Analytical Digest of plasmids using Fermentas FastDigest Enzymes

• Note: This is a small scale digest, which serves to check the identity of the plasmid and parts.

1. Mix:
   • up to 200 ng plasmid DNA (typically ~1-2 uL of a mini-prep)
   • 1 µL 10x FastDigest buffer
   • distilled water to 10 µL total volume
   • 0.5 µL enzyme 1 (1 Fast Digest unit/µL), typically either EcoRI or XbaI
   • 0.5 µL enzyme 2 (1 Fast Digest unit/µL), typically either SpeI or PstI
2. Incubate at least 5 minutes in a metal block at 37 °C.
3. Add 2.5 µL 5x DNA loading buffer and run on appropriate percentage agarose gel.

Protocol 2: Analytical Digest of plasmids using NEB Enzymes

1. Use the double digest finder (on the NEB website)to identify the best NEBuffer, the recommended incubation temperature, and whether addition of BSA is recommended.
2. Mix:
   • up to 200 ng plasmid DNA (typically ~1-2 uL of a mini-prep)
   • 1 µL 10x NEB buffer
   • if suggested, 1 µL 10x BSA
   • distilled water to 10 µL total volume
   • 0.5 µL enzyme 1 (1 Fast Digest unit/µL), typically either EcoRI or XbaI
   • 0.5 µL enzyme 2 (1 Fast Digest unit/µL), typically either SpeI or PstI
3. Incubate at least 30 minutes in a metal block at recommended temperature.
4. Add 2.5 µL 5x DNA loading buffer and run on appropriate percentage agarose gel.