Corum:Digestion: Difference between revisions
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Sean P Corum (talk | contribs) 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 # (#...) |
Sean P Corum (talk | contribs) |
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* X μL DNA template | * X μL DNA template | ||
* NEB Digestion Buffer | * 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 + | # 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 | *'''SC 13:48, 26 July 2012 (EDT)''': | ||
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | <!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | ||
Latest revision as of 10:48, 26 July 2012
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
- 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 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)
- 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.
- 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!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- 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, books, and websites
Contact
or instead, discuss this protocol.
Digital Signature
- SC 13:48, 26 July 2012 (EDT):
