Corum:Digestion

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m (Procedure)
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# Determine reaction volume V by computing V = [(X + 1)/0.8] μL (single digest) or V = [(X + 2)/0.8] μL (double digest).
# Determine reaction volume V by computing V = [(X + 1)/0.8] μL (single digest) or V = [(X + 2)/0.8] μL (double digest).
# To DNA sample, add
# To DNA sample, add
-
#* 0.1V 10X Buffer Y
+
#* 0.1V 10X Buffer Y (Y = 1, 2, 3, or 4)
#* 0.1V 10mg/ml BSA
#* 0.1V 10mg/ml BSA
#* 1 μL restriction enzyme 1
#* 1 μL restriction enzyme 1
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# Incubate 3 hr to overnight at reaction temperature given by NEB (standard = 37 °C).
# Incubate 3 hr to overnight at reaction temperature given by NEB (standard = 37 °C).
# Optional: Heat inactivate restriction enzyme(s) by incubation 80 °C 20 min. Not necessary when digestion product is going to be purified or gel extracted, as these procedures remove the enzymes.
# Optional: Heat inactivate restriction enzyme(s) by incubation 80 °C 20 min. Not necessary when digestion product is going to be purified or gel extracted, as these procedures remove the enzymes.
 +
 +
*Example: for double digestions of 22 μL plasmid DNA, V = (22 + 2)/0.8 μL = 30 μL, and the components are:
 +
** 22 μL plasmid DNA
 +
** 3 μL 10X NEB Buffer Y
 +
** 3 μL 10mg/ml BSA
 +
** 1 μL restriction enzyme 1
 +
** 1 μL restriction enzyme 2
==Notes==
==Notes==

Revision as of 12:43, 26 July 2012

Contents

Overview

Standard DNA single or double digest of variable volume V using NEB restriction enzymes.

Materials

  • X μL DNA template
  • NEB Digestion Buffer # (# = 1, 2, 3, or 4)
  • 10mg/ml BSA

Procedure

  1. Determine compatibility and reaction conditions using Double Digest Finder (double digest only).
  2. Determine reaction volume V by computing V = [(X + 1)/0.8] μL (single digest) or V = [(X + 2)/0.8] μL (double digest).
  3. To DNA sample, add
    • 0.1V 10X Buffer Y (Y = 1, 2, 3, or 4)
    • 0.1V 10mg/ml BSA
    • 1 μL restriction enzyme 1
    • 1 μL restriction enzyme 2 (double digest only)
  4. Incubate 3 hr to overnight at reaction temperature given by NEB (standard = 37 °C).
  5. Optional: Heat inactivate restriction enzyme(s) by incubation 80 °C 20 min. Not necessary when digestion product is going to be purified or gel extracted, as these procedures remove the enzymes.
  • Example: for double digestions of 22 μL plasmid DNA, V = (22 + 2)/0.8 μL = 30 μL, and the components are:
    • 22 μL plasmid DNA
    • 3 μL 10X NEB Buffer Y
    • 3 μL 10mg/ml BSA
    • 1 μL restriction enzyme 1
    • 1 μL restriction enzyme 2

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Relevant papers, books, and websites

  1. New England Biolabs

Contact

or instead, discuss this protocol.

Digital Signature

  • SC 14:15, 29 June 2012 (EDT):
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