Corum:Digestion

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 Revision as of 13:44, 26 July 2012 (view source)m (→Procedure)← Previous diff Revision as of 13:45, 26 July 2012 (view source)m (→Procedure)Next diff → Line 14: Line 14: # 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 (Y = 1, 2, 3, or 4) + #* 0.1V 10X NEB 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

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 NEB 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 lab standard 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!

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References

Relevant papers, books, and websites

1. New England Biolabs

Contact

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Digital Signature

• SC 14:15, 29 June 2012 (EDT):