NanoBio:Old Restriction Digest: Difference between revisions
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==Digestion and De-Phosphorylation of a Plasmid using NEB enzymes== | ==Digestion and De-Phosphorylation of a Plasmid using NEB enzymes== | ||
# Use the double digest finder ([ | # Use the double digest finder ([https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder on the NEB website])to identify the best NEBuffer, the recommended incubation temperature, and whether addition of BSA is recommended. | ||
# Mix: | # Mix, but do not vortex: | ||
#*1 µg plasmid | #*1 µg plasmid | ||
#*5 µL 10x NEBuffer | #*5 µL 10x NEBuffer | ||
Line 16: | Line 16: | ||
# Incubate for 15 minutes at 37°C for 5´ extensions or blunt-ends, 60 minutes for 3´ extensions. | # Incubate for 15 minutes at 37°C for 5´ extensions or blunt-ends, 60 minutes for 3´ extensions. | ||
# Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 65°C. | # Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 65°C. | ||
# Do not purify the digested, de-phosphorylated vector before use in a ligation. Use as is. | |||
==Digestion of PCR product: Procedure using NEB Enzymes== | |||
==Digestion of PCR product: | |||
# Mix: | # Mix: | ||
#*All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL) | #*All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL) | ||
#* | #*5 µL 10x buffer (see [https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder NEB website] for optimal double digest buffer choices) | ||
#*5 µL 10x BSA, if needed | |||
#* | #*1 µL enzyme 1 (20 units/µL) | ||
#* | #*1 µL enzyme 2 (20 units/µL) | ||
#*distilled water to | #*distilled water to 50 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. | #*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. | # Incubate at least an hour (better overnight) at 37 °C. |
Latest revision as of 12:50, 21 February 2013
Digestion and De-Phosphorylation of a Plasmid using NEB enzymes
- Use the double digest finder (on the NEB website)to identify the best NEBuffer, the recommended incubation temperature, and whether addition of BSA is recommended.
- Mix, but do not vortex:
- 1 µg plasmid
- 5 µL 10x NEBuffer
- 5 µL 10 x BSA, if needed
- 1 µL enzyme 1 (10 units/µL)
- 1 µL enzyme 2 (10 units/µL)
- distilled water to 50 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: In order to scale-up this procedure use 5-10 Units of enzyme for each 1 µg plasmid.
- Incubate at recommended temperature for 1 h.
- Add, then mix:
- 5 µL 10X Antarctic Phosphatase Reaction Buffer.
- 1 µl Antarctic Phosphatase (5 units)
- Incubate for 15 minutes at 37°C for 5´ extensions or blunt-ends, 60 minutes for 3´ extensions.
- Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 65°C.
- Do not purify the digested, de-phosphorylated vector before use in a ligation. Use as is.
Digestion of PCR product: Procedure using NEB Enzymes
- Mix:
- All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)
- 5 µL 10x buffer (see NEB website for optimal double digest buffer choices)
- 5 µL 10x BSA, if needed
- 1 µL enzyme 1 (20 units/µL)
- 1 µL enzyme 2 (20 units/µL)
- distilled water to 50 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 using NEB Enzymes
- 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.
- PCR purify with Qiagen
- CAjoF 20:30, 19 February 2013 (EST):