# Corum:Digestion

(Difference between revisions)
 Revision as of 14:15, 29 June 2012 (view source)m (New page: ==Overview== Standard DNA single or double digest of variable volume V using [http://www.neb.com NEB] restriction enzymes. ==Materials== * X μL DNA template * NEB Digestion Buffer # (#...)← Previous diff Current revision (13:48, 26 July 2012) (view source)m (→Digital Signature) (7 intermediate revisions not shown.) Line 6: Line 6: * X μL DNA template * X μL DNA template - * NEB Digestion Buffer # (# = 1, 2, 3, or 4) + * 10X NEB Digestion Buffer Y (Y = 1, 2, 3, or 4) * 10mg/ml BSA * 10mg/ml BSA 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 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 # To DNA sample, add - #* 0.1V 10X Buffer Y + #* 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 Line 20: Line 20: # 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. + + + *Lab standard double digestion uses X = 22 μL plasmid DNA, so V = (22 + 2)/0.8 μL = 30 μL. + ** 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== Line 41: Line 49: ==Digital Signature== ==Digital Signature== - *'''SC 14:15, 29 June 2012 (EDT)''': + *'''SC 13:48, 26 July 2012 (EDT)''':

## Overview

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

## Materials

• X μL DNA template
• 10X NEB Digestion Buffer Y (Y = 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.

• Lab standard double digestion uses X = 22 μL plasmid DNA, so V = (22 + 2)/0.8 μL = 30 μL.
• 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.

## References

Relevant papers, books, and websites

1. New England Biolabs

## Contact

or instead, discuss this protocol.

## Digital Signature

• SC 13:48, 26 July 2012 (EDT):