Corum:Digestion: Difference between revisions

Revision as of 13:08, 29 June 2012

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

Determine compatibility and reaction conditions using Double Digest Finder (double digest only).
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
- 0.1V 10X Buffer Y
- 0.1V 10mg/ml BSA
- 1 μL restriction enzyme 1
- 1 μL restriction enzyme 2 (double digest only)
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.

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@@ Line 12: / Line 12: @@
 # Determine compatibility and reaction conditions using [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp#.T-3vVXixm19NEB Double Digest Finder] (double digest only).
-# Determine reaction volume V by computing V = [(X + 1)/0.8] μL (single digest) or V = [(X + 1)/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
 #* 0.1V 10X Buffer Y