Silver: Restriction Digest: Difference between revisions
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#*700 ng BioBrick vector (BBa_V0002 or BBa_V0100) | #*700 ng BioBrick vector (BBa_V0002 or BBa_V0100) | ||
#*1 µL 10 x BSA | #*1 µL 10 x BSA | ||
#*1 µL | #*1 µL 10x buffer (see [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp? NEB website] for optimal double digest buffer choices) | ||
#*0.2 µL enzyme 1 (20 units/µL) | #*0.2 µL enzyme 1 (20 units/µL) | ||
#*0.2 µL enzyme 2 (20 units/µL) | #*0.2 µL enzyme 2 (20 units/µL) | ||
Revision as of 11:28, 31 October 2005
Digestion of PCR product
- 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.
- Incubate at least an hour (better overnight) at 37 °C.
- Purify digested insert using PCR product purification kit.
Digestion of BioBrick vector
- Mix:
- 700 ng BioBrick 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.
- Incubate overnight at 37 °C.
- The next morning, add 0.1 µL CIP (10 units/µL) and incubate for 1 hr. at 37 °C.
