NanoBio: Restriction Digest

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(New page: ==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 [http://www...)
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==Digestion & de-phosphorylation of plasmids using Fermentas FastDigest Enzymes==
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*Note from Caroline: I think Fermentas's Fast Digest Enzymes are awesome!
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# Mix:
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#*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)
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#*3 µL 10x FastDigest buffer
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#*distilled water to 30 µL total volume
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#*1 µL enzyme 1 (1 Fast Digest unit/µL)
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#*1 µL enzyme 2 (1 Fast Digest unit/µL)
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#*1 µL calf intestinal alkaline phosphatase (CIP) (10 units/µL)
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# Incubate at least an hour in a metal block at 37 °C.
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# Purify digested insert using PCR product purification kit.
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==Digestion of PCR product using NEB Enzymes==
==Digestion of PCR product using NEB Enzymes==
# Mix:  
# Mix:  

Revision as of 14:48, 26 January 2008

Digestion & de-phosphorylation of plasmids using Fermentas FastDigest Enzymes

  • Note from Caroline: I think Fermentas's Fast Digest Enzymes are awesome!
  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)
  1. Incubate at least an hour in a metal block at 37 °C.
  2. Purify digested insert using PCR product purification kit.

Digestion of PCR product using NEB Enzymes

  1. 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.
  2. Incubate at least an hour (better overnight) at 37 °C.
  3. Purify digested insert using PCR product purification kit.

Digestion of Biofusion vector: Old Procedure

  1. 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.
  2. Incubate overnight at 37 °C.
  3. The next morning, add 0.1 µL CIP (10 units/µL) and incubate for 1 hr. at 37 °C.
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