Prbbbb:fusion assembly v1: Difference between revisions
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m (New page: Back to all protocols ==Overview== ==Materials== *100 ul + PCR tubes *Phusion HotStart Polymerase 2 U/ul *Phusion HF Buffer 5x *dNTP mix 10mM each nucleotide ...) |
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==Materials== | ==Materials== | ||
* | *enzymes: AgeI, [[EcoRI]], [[PstI]], NgoMIV (=NgoMI), [[T4_DNA_Ligase]] | ||
*[[ | *[[NEB_buffers]]: NEBuffer EcoRI, NEBuffer 1, NEBuffer 4, T4 DNA Ligase buffer | ||
*NEB BSA 100x concentrated | |||
* | |||
*ddH2O | *ddH2O | ||
*linear vector backbone DNA from [[Prbbbb:vector_pcr]]; concentration: '''25 ng/µl''' | |||
*linear vector backbone DNA from [[Prbbbb:vector_pcr]] | |||
**[http://brickit.crg.es/registry/vector/11/ pSB1AC3F (CRG)] -- [http://partsregistry.org/Part:BBa_J18901 BBa_J18901] | **[http://brickit.crg.es/registry/vector/11/ pSB1AC3F (CRG)] -- [http://partsregistry.org/Part:BBa_J18901 BBa_J18901] | ||
**[http://brickit.crg.es/registry/vector/12/ pSB1AK3F (CRG)] -- [http://partsregistry.org/Part:BBa_J18901 BBa_J18902] | **[http://brickit.crg.es/registry/vector/12/ pSB1AK3F (CRG)] -- [http://partsregistry.org/Part:BBa_J18901 BBa_J18902] | ||
**[http://brickit.crg.es/registry/vector/13/ pSB1AT3F (CRG)] -- [http://partsregistry.org/Part:BBa_J18901 BBa_J18903] | **[http://brickit.crg.es/registry/vector/13/ pSB1AT3F (CRG)] -- [http://partsregistry.org/Part:BBa_J18901 BBa_J18903] | ||
*DNA Biobrick(s) A (first part), Biobrick(s) B (second part); concentration '''50 ng/µl''' | |||
==Procedure== | ==Procedure== | ||
Revision as of 11:23, 11 February 2009
Overview
Materials
- enzymes: AgeI, EcoRI, PstI, NgoMIV (=NgoMI), T4_DNA_Ligase
- NEB_buffers: NEBuffer EcoRI, NEBuffer 1, NEBuffer 4, T4 DNA Ligase buffer
- NEB BSA 100x concentrated
- ddH2O
- linear vector backbone DNA from Prbbbb:vector_pcr; concentration: 25 ng/µl
- DNA Biobrick(s) A (first part), Biobrick(s) B (second part); concentration 50 ng/µl
Procedure
setup PCR reaction
| µl, single reaction | 3.5xMaster | 10xMaster | |
|---|---|---|---|
| H2O | 76µl | 266 | 760 |
| 5x HF Buffer | 20µl | 70 | 200 |
| 10mM dNTP | 2µl | 7 | 20 |
| rg0301 100 µM | 0.5µl | 1.75 | 5 |
| rg0302 100 µM | 0.5µl | 1.75 | 5 |
| Phusion | 1µl | 3.5 | 10 |
| template DNA | 0.1µl | -- | -- |
PCR program
- 30"@98C;
- 35x (10"@98°C; 15"@68C; 1'30"@72C);
- 10'@72C;
- inf. 4C
Post-Processing
- add 1µl DpnI, incubate for 1h @ 37°C
- purify with PCR purification kit
- elute in water **not** elution buffer
- dilute to standard concentration: 50ng/µl
Notes
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References
Example reference
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