Silver: Restriction Digest

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==Digestion of PCR product==
==Digestion of PCR product==
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For FastDigest enzymes:
 +
#Mix:
 +
#*All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)
 +
#*3.5 µL 10X Buffer
 +
#*0.5 µL of each enzyme you want to use
 +
#*Water up to 35 µL
 +
#Incubate at 37°C in a waterbath for 5-30 minutes (slightly longer if using an air incubator)
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#Purify digested PCR product using PCR product purification kit
 +
 +
 +
For NEB enzymes:
# Mix:  
# Mix:  
#*All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)  
#*All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)  
Line 18: Line 29:
#*Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.
#*Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.
# Incubate at least an hour (better overnight) at 37 °C.
# Incubate at least an hour (better overnight) at 37 °C.
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# Purify digested insert using PCR product purification kit.
+
# Purify digested PCR product using PCR product purification kit.
==Digestion of BioBrick vector: Recommended Procedure==
==Digestion of BioBrick vector: Recommended Procedure==

Revision as of 10:15, 29 June 2010

Contents

Fermentas FastDigest Protocol

  1. Mix:
    • 2 µL 10X Buffer
    • up to 200 ng DNA
    • 0.5 µL of each enzyme you want to use
    • Water up to 20 µL
  2. Incubate at 37°C in a waterbath for 5-30 minutes (slightly longer if using an air incubator)
  3. Purify digested insert using gel extraction or PCR product purification kit

Digestion of PCR product

For FastDigest enzymes:

  1. Mix:
    • All of PCR product DNA (~28 µL if PCR purification was eluted in 30 µL)
    • 3.5 µL 10X Buffer
    • 0.5 µL of each enzyme you want to use
    • Water up to 35 µL
  2. Incubate at 37°C in a waterbath for 5-30 minutes (slightly longer if using an air incubator)
  3. Purify digested PCR product using PCR product purification kit


For 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 PCR product using PCR product purification kit.

Digestion of BioBrick vector: Recommended Procedure

  1. Mix:
    • 700 ng BioBrick 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.

BioCoder version

Following is the Silver:Restriction digest protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.Vaishnavi

Text Output

Silver:Restriction digest protocol

Source Code

Silver:Restriction digest protocol - source code

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