NanoBio:Old Restriction Digest

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Digestion and De-Phosphorylation of a Plasmid 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, 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
    • 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.
  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.

Digestion of PCR product: Procedure using NEB Enzymes

  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
    • Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.
  2. Incubate at least an hour (better overnight) at 37 °C.
  3. Purify digested insert using PCR product purification kit.

Digestion of Biofusion vector: Old Procedure using NEB Enzymes

  1. Mix:
    • 700 ng Biofusion vector (BBa_V0002 or BBa_V0100)
    • 2 µL 10 x BSA
    • 2 µL 10x buffer (see NEB website for optimal double digest buffer choices)
    • 0.3 µL enzyme 1 (20 units/µL)
    • 0.3 µL enzyme 2 (20 units/µL)
    • 0.4 uL calf alkaline phosphatase (CIP)
    • distilled water to 20 µL total volume
    • Note: To keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 10% of the total reaction volume.
  2. Incubate overnight at 37 °C.
  3. PCR purify with Qiagen
  • CAjoF 20:30, 19 February 2013 (EST):
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