Corum:digest: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Sean P Corum (talk | contribs) mNo edit summary |
Sean P Corum (talk | contribs) mNo edit summary |
||
| Line 2: | Line 2: | ||
==Overview== | ==Overview== | ||
Standard DNA digest protocol | Standard DNA digest protocol using [http://www.neb.com | NEB] restriction enzymes. | ||
==Materials== | ==Materials== | ||
| Line 15: | Line 15: | ||
==Procedure== | ==Procedure== | ||
# Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com|NEB]. | # Find appropriate enzymes(s), associated buffer, reaction temperature from [http://www.neb.com | NEB]. | ||
# Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V. | # Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V. | ||
# Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V. | # Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V. | ||
| Line 33: | Line 33: | ||
<!-- If this protocol has papers or books associated with it, list those references here.--> | <!-- If this protocol has papers or books associated with it, list those references here.--> | ||
<!-- Try the [[Template:FormatRef|FormatRef template]]--> | <!-- Try the [[Template:FormatRef|FormatRef template]]--> | ||
# | #[http://www.neb.com | New England Biolabs] | ||
==Contact== | ==Contact== | ||
Revision as of 11:49, 19 June 2012
- SC 14:47, 19 June 2012 (EDT):
Overview
Standard DNA digest protocol using | NEB restriction enzymes.
Materials
For a DNA digestion of total volume V (in μL):
- ~0.8V DNA
- 0.1V 10X digestion buffer (1, 2, 3, or 4; NEB)
- 0.1V 10mg/ml BSA
- 1 μL restriction enzyme 1
- 1 μL restriction enzyme 2 (optional, for double digestion)
Procedure
- Find appropriate enzymes(s), associated buffer, reaction temperature from | NEB.
- Calculate the total volume of the reaction by taking the DNA volume + 1(2) μL for a single (double) digestion reaction. Divide this number by 0.8 to get the total reaction volume, V.
- Calculate the volume of 10mg/ml BSA and 10X digestion buffer, which are both 0.1V.
- Add the reaction components together in order listed above. Mix gently and spin.
- Incubate at reaction temperature for 3 hr to overnight.
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 and books
Contact
or instead, discuss this protocol.
