NanoBio: Restriction Digest: Difference between revisions
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==Materials== | |||
*Fermentas FastDigest enzymes & 10x FastDigest buffer | |||
**EcoRI catalog #FD0274, XbaI catalog #FD0684, SpeI catalog #FD1254, PstI catalog #FD0614 | |||
*Calf alkaline phosphatase, Fisher catalog #50811712(aka NEB catalog #M0290S) or Fermentas catalog #EF0341 | |||
*Qiagen PCR purification kit | |||
==Digestion & de-phosphorylation of plasmids using Fermentas FastDigest Enzymes== | ==Digestion & de-phosphorylation of plasmids using Fermentas FastDigest Enzymes== | ||
# Mix: | # Mix: |
Revision as of 19:36, 30 January 2008
Materials
- Fermentas FastDigest enzymes & 10x FastDigest buffer
- EcoRI catalog #FD0274, XbaI catalog #FD0684, SpeI catalog #FD1254, PstI catalog #FD0614
- Calf alkaline phosphatase, Fisher catalog #50811712(aka NEB catalog #M0290S) or Fermentas catalog #EF0341
- Qiagen PCR purification kit
Digestion & de-phosphorylation of plasmids using Fermentas FastDigest Enzymes
- 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)
- Incubate at least an hour in a metal block at 37 °C.
- Purify plasmid using Qiagen's PCR product purification kit.
Digestion of plasmids using Fermentas FastDigest Enzymes
- 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)
- Incubate at least 5 minutes in a metal block at 37 °C.
- Purify digested insert & plasmid using PCR product purification kit.
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)
- 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.