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Protocol/13
From 2010.igem.org
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| - | Protocol 13: Vector | + | Protocol 13: Vector Dephosphorylation |
| - | + | ||
| + | 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- | + | * Add 1/10 volume of 10x antarctic phosphatase reaction buffer to 1-5ug of DNA cut with any restriction endonuclease in any buffer. |
| - | * Add | + | * Add 1ul of Antarctic phosphatase and mix. |
| - | * Incubate 5 | + | * Incubate 5 minutes at 37<sup>o</sup>C. |
| - | * Heat inactivate for 5 minutes at 65 C | + | * Heat inactivate for 5 minutes at 65<sup>o</sup>C. |
| - | * Proceed with ligation | + | * 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. | ||
| - | |||
| - | |||
[[Team:Alberta/Notebook/protocols| Back]] | [[Team:Alberta/Notebook/protocols| Back]] | ||
{{Team:Alberta/endMainContent}} | {{Team:Alberta/endMainContent}} | ||
Revision as of 01:37, 26 October 2010
Protocol 13: 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.
