"
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...) |
|||
| (3 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
{{Team:Alberta/Head}} | {{Team:Alberta/Head}} | ||
| - | {{Team:Alberta/navbar| | + | {{Team:Alberta/navbar|notebook=selected}} |
| + | |||
| + | {{Team:Alberta/beginLeftSideBar|toc=NO}} | ||
| + | |||
| + | |||
| + | ==General Protocols== | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/invitro_biobyte_assembly | In Vitro BioByte Assembly]] | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/LB | LB Plates and Broth]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/transformations |Transformations]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/overnight |5mL Overnight ]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/glycerol | Glycerol Stock ]] | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/miniprep | Plasmid Miniprep ]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/digest | Restriction Digest ]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/vector_dephos | Vector Dephosphorylation]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/ligation | Ligation ]] | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/agarose_gel | Agarose Gel Electrophoresis ]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/gel_extraction | Gel Extraction ]] | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/pcr | PCR]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/colony_pcr | Colony PCR ]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/pcr_purification | PCR Purification ]] | ||
| + | ----------------------------- | ||
| + | *[[Team:Alberta/Notebook/protocols/labelling | Sample Labelling Conventions]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/sequencing | Fluorescent Sequencing Reaction]] | ||
| + | |||
| + | *[[Team:Alberta/Notebook/protocols/primer_design | Primer Design]] | ||
| + | ----------------------------- | ||
| + | |||
| + | |||
| + | {{Team:Alberta/endMainContent}} | ||
| + | |||
| - | |||
{{Team:Alberta/endLeftSideBar}} | {{Team:Alberta/endLeftSideBar}} | ||
| Line 11: | Line 54: | ||
{{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 71: | ||
| - | 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. | ||
* It chews off 5' phosphates, but it also keeps chewing on the DNA ends, reducing total transformation efficiency. | * 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. | * Try without dephosphorylation first, and minimize exposure to phosphatase if you must resort to using it. | ||
| - | + | * See also Sambrook. | |
| - | |||
{{Team:Alberta/endMainContent}} | {{Team:Alberta/endMainContent}} | ||
Latest revision as of 02:35, 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.
