NanoBio:Old Restriction Digest: Difference between revisions
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(New page: ==Digestion of PCR product using NEB Enzymes== # 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 [http://www...) |
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| Line 14: | Line 14: | ||
# Mix: | # Mix: | ||
#*700 ng Biofusion vector (BBa_V0002 or BBa_V0100) | #*700 ng Biofusion vector (BBa_V0002 or BBa_V0100) | ||
#* | #*2 µL 10 x BSA | ||
#* | #*2 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. | #*0.3 µL enzyme 1 (20 units/µL) | ||
#*0. | #*0.3 µL enzyme 2 (20 units/µL) | ||
#*distilled water to | #*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. | #*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. | # Incubate overnight at 37 °C. | ||
# The next morning, add 0.1 µL CIP (10 units/µL) and incubate for 1 hr. at 37 °C. | # The next morning, add 0.1 µL CIP (10 units/µL) and incubate for 1 hr. at 37 °C. | ||
Revision as of 13:38, 16 April 2008
Digestion of PCR product using NEB Enzymes
- 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 Biofusion vector: Old Procedure
- 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.
- Incubate overnight at 37 °C.
- The next morning, add 0.1 µL CIP (10 units/µL) and incubate for 1 hr. at 37 °C.
