NanoBio: Restriction Digest

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*Note: This protocol details a large scale digestion, the products of which will be pcr purified and used in a ligation.
*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 product
#*2 µL 10x FastDigest buffer
#*2 µL 10x FastDigest buffer
#*distilled water to 20 µL total volume
#*distilled water to 20 µL total volume

Revision as of 13:22, 1 July 2008

Contents

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.
  1. 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)
  2. Incubate at least an hour in a metal block at 37 °C.
  3. 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.
  1. 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 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)
  2. Incubate at least 5 minutes in a metal block at 37 °C.
  3. 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.
  1. 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
  2. Incubate at least 5 minutes in a metal block at 37 °C.
  3. Add 2.5 µL 5x DNA loading buffer and run on appropriate percentage agarose gel.

Here you can find instructions for NEB Restriction Digest.

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