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Team:Alberta/Notebook/protocols/vector dephos
From 2010.igem.org
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{{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}} | ||
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* 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.
