NanoBio: Restriction Digest

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*Calf alkaline phosphatase, Fisher catalog #50811712(aka NEB catalog #M0290S) or Fermentas catalog #EF0341
*Calf alkaline phosphatase, Fisher catalog #50811712(aka NEB catalog #M0290S) or Fermentas catalog #EF0341
*Qiagen PCR purification kit
*Qiagen PCR purification kit
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==Digestion & de-phosphorylation of plasmids using Fermentas FastDigest Enzymes==
==Digestion & de-phosphorylation of plasmids using Fermentas FastDigest Enzymes==
# Mix:  
# Mix:  
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# Purify digested insert & plasmid using PCR product purification kit.
# Purify digested insert & plasmid using PCR product purification kit.
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==Digestion of PCR product using NEB Enzymes==
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Here you can find instructions for [[NanoBio:Old Restriction Digest|NEB Restriction Digest]].
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# Mix:
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#*All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)
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#*3.5 µL 10x BSA
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#*3.5 µL 10x buffer (see [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp? NEB website] for optimal double digest buffer choices) 
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#*0.2 µL enzyme 1 (20 units/µL)
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#*0.2 µL enzyme 2 (20 units/µL)
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#*distilled water to 35 µL total volume
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#*Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.
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# Incubate at least an hour (better overnight) at 37 °C.
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# Purify digested insert using PCR product purification kit.
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==Digestion of Biofusion vector: Old Procedure==
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# Mix:
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#*700 ng Biofusion vector (BBa_V0002 or BBa_V0100)
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#*1 µL 10 x BSA
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#*1 µL 10x buffer (see [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp? NEB website] for optimal double digest buffer choices)
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#*0.2 µL enzyme 1 (20 units/µL)
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#*0.2 µL enzyme 2 (20 units/µL)
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#*distilled water to 10 µL total volume
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#*Note: To keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 10% of the total reaction volume.
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# Incubate overnight at 37 °C.
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# The next morning, add 0.1 µL CIP (10 units/µL) and incubate for 1 hr. at 37 °C.
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Revision as of 21:39, 30 January 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

Digestion & de-phosphorylation of plasmids using Fermentas FastDigest Enzymes

  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.

Digestion of plasmids using Fermentas FastDigest Enzymes

  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)
    • 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.

Here you can find instructions for NEB Restriction Digest.

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