Corum:Digestion

(Difference between revisions)
 Revision as of 16:08, 29 June 2012 (view source)m (→Procedure)← Previous diff Current revision (13:48, 26 July 2012) (view source)m (→Digital Signature) (6 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 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 + #* 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).
• 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