NanoBio: Restriction Digest

Digestion & de-phosphorylation of plasmids using Fermentas FastDigest 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.

Digestion of plasmids using Fermentas FastDigest 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)
   • 2 µL 10x FastDigest buffer
   • distilled water to 20 µL total volume
   • 1 µL enzyme 1 (1 Fast Digest unit/µL)
   • 1 µL enzyme 2 (1 Fast Digest unit/µL)
2. Incubate at least 5 minutes in a metal block at 37 °C.
3. Purify digested insert & plasmid using PCR product purification kit.

Digestion of PCR product using NEB Enzymes

1. Mix:
   • All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)
   • 3.5 µL 10x BSA
   • 3.5 µL 10x buffer (see NEB website for optimal double digest buffer choices)
   • 0.2 µL enzyme 1 (20 units/µL)
   • 0.2 µL enzyme 2 (20 units/µL)
   • distilled water to 35 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

1. Mix:
   • 700 ng Biofusion vector (BBa_V0002 or BBa_V0100)
   • 1 µL 10 x BSA
   • 1 µL 10x buffer (see NEB website for optimal double digest buffer choices)
   • 0.2 µL enzyme 1 (20 units/µL)
   • 0.2 µL enzyme 2 (20 units/µL)
   • distilled water to 10 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. The next morning, add 0.1 µL CIP (10 units/µL) and incubate for 1 hr. at 37 °C.