NanoBio:Old Restriction Digest

Digestion of PCR product using NEB Enzymes

 * 1) Mix:
 * 2) *All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)
 * 3) *3.5 µL 10x BSA
 * 4) *3.5 µL 10x buffer (see NEB website for optimal double digest buffer choices)
 * 5) *0.2 µL enzyme 1 (20 units/µL)
 * 6) *0.2 µL enzyme 2 (20 units/µL)
 * 7) *distilled water to 35 µL total volume
 * 8) *Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.
 * 9) Incubate at least an hour (better overnight) at 37 °C.
 * 10) Purify digested insert using PCR product purification kit.

Digestion of Biofusion vector: Old Procedure

 * 1) Mix:
 * 2) *700 ng Biofusion vector (BBa_V0002 or BBa_V0100)
 * 3) *2 µL 10 x BSA
 * 4) *2 µL 10x buffer (see NEB website for optimal double digest buffer choices)
 * 5) *0.3 µL enzyme 1 (20 units/µL)
 * 6) *0.3 µL enzyme 2 (20 units/µL)
 * 7) *0.4 uL calf alkaline phosphatase (CIP)
 * 8) *distilled water to 20 µL total volume
 * 9) *Note: To keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 10% of the total reaction volume.
 * 10) Incubate overnight at 37 °C.
 * 11) PCR purify with Qiagen