Corum:digest

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m
m
Line 2: Line 2:
==Overview==
==Overview==
-
Standard DNA digest protocol (NEB restriction enzymes).
+
Standard DNA digest protocol using [http://www.neb.com | NEB] restriction enzymes.
==Materials==
==Materials==
Line 15: Line 15:
==Procedure==
==Procedure==
-
# Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com|NEB].
+
# Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com | NEB].
# Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.
# Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.
# Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
# Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
Line 33: Line 33:
<!-- If this protocol has papers or books associated with it, list those references here.-->
<!-- If this protocol has papers or books associated with it, list those references here.-->
<!-- Try the [[Template:FormatRef|FormatRef template]]-->
<!-- Try the [[Template:FormatRef|FormatRef template]]-->
-
#{{FormatRef|Sambrook, J and Russell, DW|2001|Molecular Cloning: A Laboratory Manual (Volume II)| | |Cold Spring Harbor Laboratory Press}} ISBN 0879695773
+
#[http://www.neb.com | New England Biolabs]
==Contact==
==Contact==

Revision as of 14:49, 19 June 2012

  • SC 14:47, 19 June 2012 (EDT):

Contents

Overview

Standard DNA digest protocol using | NEB restriction enzymes.

Materials

For a DNA digestion of total volume V (in μL):

  • ~0.8V DNA
  • 0.1V 10X digestion buffer (1, 2, 3, or 4; NEB)
  • 0.1V 10mg/ml BSA
  • 1 μL restriction enzyme 1
  • 1 μL restriction enzyme 2 (optional, for double digestion)

Procedure

  1. Find appropriate enzymes(s), associated buffer, reaction temperature from | NEB.
  2. Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.
  3. Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
  4. Add the reaction components together in order listed above. Mix gently and spin.
  5. Incubate at reaction temperature for 3 hr to overnight.

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 and books

  1. | New England Biolabs

Contact

or instead, discuss this protocol.

Personal tools