NanoBio: Restriction Digest

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(Preparative Digestion of plasmids using Fermentas FastDigest Enzymes)
 
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==Digestion & de-phosphorylation of plasmids using Fermentas FastDigest Enzymes==
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==Materials==
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*Note from Caroline: I think Fermentas's Fast Digest Enzymes are awesome!
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*Fermentas FastDigest enzymes & 10x FastDigest buffer
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**EcoRI catalog #FD0274, XbaI catalog #FD0684, SpeI catalog #FD1254, PstI catalog #FD0614
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*Calf alkaline phosphatase, Fisher catalog #50811712(aka NEB catalog #M0290S) or Fermentas catalog #EF0341
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*Qiagen PCR purification kit
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==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)  
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#*1 µL enzyme 2 (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 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.
# 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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# Purify plasmid using Qiagen's PCR product purification kit.
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==Digestion of PCR product using NEB Enzymes==
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==Preparative Digestion of plasmids using Fermentas FastDigest Enzymes==
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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.
# Mix:  
# Mix:  
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#*All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)
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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) '''OR''' up to 0.2 μg pcr-purified PCR product
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#*3.5 µL 10x BSA
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#*2 µL 10x FastDigest buffer
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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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#*distilled water to 20 µL total volume
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#*0.2 µL enzyme 1 (20 units/µL)
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#*1 µL enzyme 1 (1 Fast Digest unit/µL)
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#*0.2 µL enzyme 2 (20 units/µL)
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#*1 µL enzyme 2 (1 Fast Digest unit/µL)
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#*distilled water to 35 µL total volume
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# Incubate at least 5 minutes in a metal block at 37 °C.
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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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# Purify digested insert & plasmid using PCR product purification kit.
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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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==Analytical Digest of plasmids using Fermentas FastDigest Enzymes==
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# Mix:  
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*Note: This is a small scale digest, which serves to check the identity of the plasmid and parts.
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#*700 ng Biofusion vector (BBa_V0002 or BBa_V0100)  
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#Mix:
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#*1 µL 10 x BSA
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#*up to 200 ng plasmid DNA (typically ~1-2 uL of a mini-prep)
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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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#*1 µL 10x FastDigest buffer
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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
#*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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#*0.5 µL enzyme 1 (1 Fast Digest unit/µL), typically either EcoRI or XbaI
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# Incubate overnight at 37 °C.
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#*0.5 µL enzyme 2 (1 Fast Digest unit/µL), typically either SpeI or PstI
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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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#Incubate at least 5 minutes in a metal block at 37 °C.
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#Add 2.5 µL 5x DNA loading buffer and run on appropriate percentage agarose gel.
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== ==
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Here you can find instructions for [[NanoBio:Old Restriction Digest|NEB Restriction Digest]].<br>
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Return to [[NanoBio:Protocols|Protocols]]

Current revision

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-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)
  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.

Return to Protocols

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