NanoBio:Old Restriction Digest: Difference between revisions
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==Digestion of PCR product using NEB Enzymes== | |||
==Digestion and De-Phosphorylation using NEB enzymes== | |||
# Use double digest finder ([http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp? NEB website])to identify best NEBuffer, the recommended incubation temperature, and whether BSA is recommended. | |||
# Mix: | |||
#*1 µg plasmid | |||
#*5 µL 10x buffer | |||
#*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 recommended temperature for 1 h °C. | |||
# Add 5 µL volume of 10X Antarctic Phosphatase Reaction Buffer. | |||
# Add 1 µl of Antarctic Phosphatase (5 units) and mix. | |||
# 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. | |||
==Digestion of PCR product: Old Procedure using NEB Enzymes== | |||
# 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) | ||
| Line 11: | Line 30: | ||
# Purify digested insert using PCR product purification kit. | # Purify digested insert using PCR product purification kit. | ||
==Digestion of Biofusion vector: Old Procedure== | ==Digestion of Biofusion vector: Old Procedure using NEB Enzymes== | ||
# Mix: | # Mix: | ||
#*700 ng Biofusion vector (BBa_V0002 or BBa_V0100) | #*700 ng Biofusion vector (BBa_V0002 or BBa_V0100) | ||
| Line 23: | Line 42: | ||
# Incubate overnight at 37 °C. | # Incubate overnight at 37 °C. | ||
# PCR purify with Qiagen | # PCR purify with Qiagen | ||
*'''[[User:CarolineAjo-Franklin|CAjoF]] 20:30, 19 February 2013 (EST)''': | |||
Revision as of 18:30, 19 February 2013
Digestion and De-Phosphorylation using NEB enzymes
- Use double digest finder (NEB website)to identify best NEBuffer, the recommended incubation temperature, and whether BSA is recommended.
- Mix:
- 1 µg plasmid
- 5 µL 10x buffer
- 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 recommended temperature for 1 h °C.
- Add 5 µL volume of 10X Antarctic Phosphatase Reaction Buffer.
- Add 1 µl of Antarctic Phosphatase (5 units) and mix.
- 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.
Digestion of PCR product: Old Procedure 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 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):
