NanoBio: Restriction Digest: Difference between revisions
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==Digestion & de-phosphorylation of plasmids using Fermentas FastDigest Enzymes== | ==Materials== | ||
*Note | *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 | |||
==Preparative Digestion & de-phosphorylation of (construction) plasmids using Fermentas FastDigest Enzymes== | |||
*Note: This protocol details a large scale digestion, the products of which will be pcr purified and used in a ligation. | |||
# Mix: | # 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) | #*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) | ||
Line 11: | Line 17: | ||
# Purify plasmid using Qiagen's PCR product purification kit. | # Purify plasmid using Qiagen's PCR product purification kit. | ||
==Digestion of plasmids using Fermentas FastDigest Enzymes== | ==Preparative Digestion of plasmids using Fermentas FastDigest Enzymes== | ||
*Note: This protocol details a large scale digestion, the products of which will be pcr purified and used in a ligation. | |||
# Mix: | # 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) | #*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) '''OR''' up to 0.2 μg pcr-purified PCR product | ||
#*2 µL 10x FastDigest buffer | #*2 µL 10x FastDigest buffer | ||
#*distilled water to | #*distilled water to 20 µL total volume | ||
#*1 µL enzyme 1 (1 Fast Digest unit/µL) | #*1 µL enzyme 1 (1 Fast Digest unit/µL) | ||
#*1 µL enzyme 2 (1 Fast Digest unit/µL) | #*1 µL enzyme 2 (1 Fast Digest unit/µL) | ||
Line 21: | Line 28: | ||
# Purify digested insert & plasmid using PCR product purification kit. | # Purify digested insert & plasmid using PCR product purification kit. | ||
== | ==Analytical Digest of plasmids using Fermentas FastDigest Enzymes== | ||
# Mix: | *Note: This is a small scale digest, which serves to check the identity of the plasmid and parts. | ||
#* | #Mix: | ||
#* | #*up to 200 ng plasmid DNA (typically ~1-2 uL of a mini-prep) | ||
#* | #*1 µL 10x FastDigest buffer | ||
#*0. | #*distilled water to 10 µL total volume | ||
#*0. | #*0.5 µL enzyme 1 (1 Fast Digest unit/µL), typically either EcoRI or XbaI | ||
#*0.5 µL enzyme 2 (1 Fast Digest unit/µL), typically either SpeI or PstI | |||
#Incubate at least 5 minutes in a metal block at 37 °C. | |||
# Incubate at least | #Add 2.5 µL 5x DNA loading buffer and run on appropriate percentage agarose gel. | ||
# | |||
== == | |||
Here you can find instructions for [[NanoBio:Old Restriction Digest|NEB Restriction Digest]].<br> | |||
Return to [[NanoBio:Protocols|Protocols]] | |||
Latest revision as of 10:31, 1 July 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
Preparative Digestion & de-phosphorylation of (construction) plasmids using Fermentas FastDigest Enzymes
- Note: This protocol details a large scale digestion, the products of which will be pcr purified and used in a ligation.
- 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.
Preparative Digestion of plasmids using Fermentas FastDigest Enzymes
- Note: This protocol details a large scale digestion, the products of which will be pcr purified and used in a ligation.
- 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) OR up to 0.2 μg pcr-purified PCR product
- 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.
Analytical Digest of plasmids using Fermentas FastDigest Enzymes
- Note: This is a small scale digest, which serves to check the identity of the plasmid and parts.
- Mix:
- up to 200 ng plasmid DNA (typically ~1-2 uL of a mini-prep)
- 1 µL 10x FastDigest buffer
- distilled water to 10 µL total volume
- 0.5 µL enzyme 1 (1 Fast Digest unit/µL), typically either EcoRI or XbaI
- 0.5 µL enzyme 2 (1 Fast Digest unit/µL), typically either SpeI or PstI
- Incubate at least 5 minutes in a metal block at 37 °C.
- Add 2.5 µL 5x DNA loading buffer and run on appropriate percentage agarose gel.
Here you can find instructions for NEB Restriction Digest.
Return to Protocols