"
Protocol/5
From 2010.igem.org
(Difference between revisions)
| Line 14: | Line 14: | ||
| - | + | Reagents: | |
* Restriction enzyme buffer | * Restriction enzyme buffer | ||
| - | ** Buffer information can be found at www.NEB.com using the NEBtools | + | ** Buffer information can be found at www.NEB.com using the NEBtools. |
* Restriction enzymes | * Restriction enzymes | ||
| - | * | + | * MilliQ water |
* DNA to be digested | * DNA to be digested | ||
| Line 35: | Line 35: | ||
**Enzyme II | **Enzyme II | ||
| - | An example of a typical 25 ul reaction would be | + | An example of a typical 25 ul reaction would be: |
{| | {| | ||
|milliQ water || 15.5 ul | |milliQ water || 15.5 ul | ||
| Line 49: | Line 49: | ||
Incubate at 37 C for one hours (longer is okay too). | Incubate at 37 C for one hours (longer is okay too). | ||
| - | |||
| - | FastDigest (by Fermentas) enzymes use a single uniform buffer, and claim to work in 5 | + | Notes: |
| + | |||
| + | FastDigest (by Fermentas) enzymes use a single uniform buffer, and claim to work in 5 minutes. | ||
| + | |||
[[Team:Alberta/Notebook/protocols| Back]] | [[Team:Alberta/Notebook/protocols| Back]] | ||
{{Team:Alberta/endMainContent}} | {{Team:Alberta/endMainContent}} | ||
Revision as of 01:08, 26 October 2010
Protocol 5: Restriction Digest
Reagents:
- Restriction enzyme buffer
- Buffer information can be found at www.NEB.com using the NEBtools.
- Restriction enzymes
- MilliQ water
- DNA to be digested
Procedure:
- Select a restriction enzyme buffer that is appropriate for BOTH of the enzymes you are using. See information sheets at front of lab for correct buffer and concentration.
- The total volume of all enzymes in the reaction should be less than 10% of the final reaction volume. Enzymes usually are supplied at 10U/ul and 1ul will be more than enough for our digests.
- Add components in the following order:
- Water
- Buffer
- Bovine Serum Albumin-BSA (if needed)
- DNA
- Enzyme I
- Enzyme II
An example of a typical 25 ul reaction would be:
| milliQ water | 15.5 ul |
| 10x NEBuffer | 2.5 ul |
| DNA (200 ng/ul) | 5.0 ul |
| XbaI | 1.0 ul |
| PstI | 1.0 ul |
Incubate at 37 C for one hours (longer is okay too).
Notes:
FastDigest (by Fermentas) enzymes use a single uniform buffer, and claim to work in 5 minutes.
