Preparative Scale Digest of PCR products and Plasmids

Goal:

To cut dsDNA using restriction enzymes for use in cloning reactions (preparative scale digest).

Important Notes:

We use both Fermentas and NEB restriction enzymes for both plasmid and pcr digests, so protocols for all four variations are below.

Protocol 1: Preparative digest and de-phosphorylation of a Plasmid using Fermentas Enzymes

    1. Mix:
      • up to 1 ug plasmid DNA (typically this is ~1/10 of the total elution volume of a mini-prep from a 5 mL overnight culture)
      • 3 µL 10x FastDigest buffer
      • distilled water to 30 µL total volume
      • 1 µL enzyme 1 (1 Fast Digest unit/µL)
      • 1 µL enzyme 2 (1 Fast Digest unit/µL)
      • 1 µL calf intestinal alkaline phosphatase (CIP) (10 units/µL)
    2. Incubate at least an hour in a metal block at 37 °C.
    3. Purify plasmid using Qiagen’s PCR product purification kit.

Protocol 2: Preparative digest of a PCR product using Fementas Enzymes

      1. Mix:
        • up to 0.2 ug pcr produtct
        • 2 µL 10x FastDigest buffer
        • distilled water to 20 µL total volume
        • 1 µL enzyme 1 (1 Fast Digest unit/µL), typically EcoRI catalog #FD0274, XbaI catalog #FD0684
        • 1 µL enzyme 2 (1 Fast Digest unit/µL), typically SpeI catalog #FD1254, PstI catalog #FD0614
      2. Incubate at least 5 minutes in a metal block at 37 °C.
      3. Calf alkaline phosphatase, Fisher catalog #50811712(aka NEB catalog #M0290S) or Fermentas catalog #EF0341
      4. Purify digested insert & plasmid using PCR product purification kit.

Protocol 2: Preparative Digest and de-phosphorylation of Plasmids using NEB Enzymes

Note: To keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 10% of the total reaction volume.
Note: In order to scale-up this procedure use 5-10 Units of enzyme for each 1 µg plasmid.

      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, but do not vortex:
        • 1 µg plasmid
        • 5 µL 10x NEBuffer
        • 5 µL 10 x BSA, if needed
        • 1 µL enzyme 1 (10 units/µL)
        • 1 µL enzyme 2 (10 units/µL)
        • distilled water to 50 µL total volume
      3. Incubate at recommended temperature for 1 h.
      4. Add, then mix:
        • 5 µL 10X Antarctic Phosphatase Reaction Buffer.
        • 1 µl Antarctic Phosphatase (5 units)
      5. Incubate for 15 minutes at 37°C for 5´ extensions or blunt-ends, 60 minutes for 3´ extensions.
      6. Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 65°C.
      7. Do not purify the digested, de-phosphorylated vector before use in a ligation. Use as is.

Protocol 3: Preparative Digest of PCR products using NEB Enzymes

Note: To keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 10% of the total reaction volume.

    1. Mix:
      • All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)
      • 5 µL 10x buffer (see NEB website for optimal double digest buffer choices)
      • 5 µL 10x BSA, if needed
      • 1 µL enzyme 1 (20 units/µL)
      • 1 µL enzyme 2 (20 units/µL)
      • distilled water to 50 µL total volume
    2. Incubate at least an hour (better overnight) at 37 °C.
    3. Purify digested insert using PCR product purification kit.

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