Prbbbb:vector pcr: Difference between revisions
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==Procedure== | ==Procedure== | ||
'''setup PCR reaction''' | |||
<table frame=box> | <table frame=box> | ||
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</table> | </table> | ||
'''PCR program''' | |||
*30"@98C; | *30"@98C; | ||
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*inf. 4C | *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== | ==Notes== | ||
Revision as of 07:39, 4 February 2009
Overview
PCR reaction to generate linearized construction vector backbones for Fusion(Freiburg)-formatted Biobrick assemblies.
Materials
- 100 ul + PCR tubes
- Phusion HotStart Polymerase 2 U/ul
- Phusion HF Buffer 5x
- dNTP mix 10mM each nucleotide
- ddH2O
- reverse prefix primer rg0301 (fusion format)
- reverse suffix primer rg0302 (fusion format)
- vector template DNA
- pSB1AC3F -- BBa_J18901
- pSB1AK3F -- BBa_J18902
- pSB1AT3F -- BBa_J18903
Procedure
setup PCR reaction
| µl, single reaction | 3.5xMaster | 10xMaster | |
|---|---|---|---|
| H2O | 76µl | 266 | 760 |
| 5x HF Buffer | 20µl | 70 | 200 |
| 10mM dNTP | 2µl | 70 | 200 |
| 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
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Relevant papers and books
- Goldbeter A and Koshland DE Jr. An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840-4. DOI:10.1073/pnas.78.11.6840 |
- JACOB F and MONOD J. Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol. 1961 Jun;3:318-56. DOI:10.1016/s0022-2836(61)80072-7 |
- ISBN:0879697164
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.
