"
Team:Alberta/Notebook/protocols/vector dephos
From 2010.igem.org
(Difference between revisions)
(New page: {{Team:Alberta/Head}} {{Team:Alberta/navbar|project=selected}} {{Team:Alberta/beginLeftSideBar}} {{Team:Alberta/endLeftSideBar}} {{Team:Alberta/beginRightSideBar}} {{Team:Alberta/endRig...) |
|||
| Line 11: | Line 11: | ||
{{Team:Alberta/beginMainContent}} | {{Team:Alberta/beginMainContent}} | ||
| - | + | ==Vector Dephosphorylation== | |
| - | Reagents: | + | ===Reagents:=== |
* Antarctic phosphatase | * Antarctic phosphatase | ||
* 10x Antarctic phosphatase buffer | * 10x Antarctic phosphatase buffer | ||
| - | Procedure: | + | ===Procedure:=== |
* Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-5ug of DNA cut with any restriction endonuclease in any buffer. | * Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-5ug of DNA cut with any restriction endonuclease in any buffer. | ||
| Line 28: | Line 28: | ||
| - | Notes: | + | ===Notes:=== |
* Vector dephosphorylation can be useful in cutting transformation background, but it is very harsh. | * Vector dephosphorylation can be useful in cutting transformation background, but it is very harsh. | ||
| Line 36: | Line 36: | ||
| - | |||
{{Team:Alberta/endMainContent}} | {{Team:Alberta/endMainContent}} | ||
Revision as of 00:43, 27 October 2010
Vector Dephosphorylation
Reagents:
- Antarctic phosphatase
- 10x Antarctic phosphatase buffer
Procedure:
- Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-5ug of DNA cut with any restriction endonuclease in any buffer.
- Add 1ul of Antarctic phosphatase and mix.
- Incubate 5 minutes at 37oC.
- Heat inactivate for 5 minutes at 65oC.
- Proceed with ligation.
Notes:
- Vector dephosphorylation can be useful in cutting transformation background, but it is very harsh.
- It chews off 5' phosphates, but it also keeps chewing on the DNA ends, reducing total transformation efficiency.
- Try without dephosphorylation first, and minimize exposure to phosphatase if you must resort to using it.
- See also Sambrook.
