Prbbbb:vector pcr: Difference between revisions
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#Step 1: *setup PCR reaction* | #Step 1: *setup PCR reaction* | ||
<table frame=box> | <table frame=box> | ||
<tr align=right> | <tr align=right> | ||
<td></td> <th width=100>µl, single reaction</th> <th width=100>3.5xMaster</th> <th width=100>10xMaster</th></tr> | <td></td> <th width=100>µl, single reaction</th> <th width=100>3.5xMaster</th> <th width=100>10xMaster</th></tr> | ||
<tr align=right> <td>H2O</td> <td>76µl</td> <td>266</td> <td>760</td> </tr> | <tr align=right> <td>H2O</td> <td>76µl</td> <td>266</td> <td>760</td> </tr> | ||
<tr align=right> <td>5x HF Buffer</td> <td>20µl</td> <td>70</td> <td>200</td> </tr> | <tr align=right> <td>5x HF Buffer</td> <td>20µl</td> <td>70</td> <td>200</td> </tr> | ||
<tr align=right> <td>10mM dNTP</td> <td>2µl</td> <td>70</td> <td>200</td> </tr> | <tr align=right> <td>10mM dNTP</td> <td>2µl</td> <td>70</td> <td>200</td> </tr> | ||
<tr><td></td></tr> | <tr><td></td></tr> | ||
<tr align=right> <td>rg0301 100 µM</td> <td>0.5µl</td> <td>1.75</td> <td>5</td> </tr> | <tr align=right> <td>rg0301 100 µM</td> <td>0.5µl</td> <td>1.75</td> <td>5</td> </tr> | ||
<tr align=right> <td>rg0302 100 µM</td> <td>0.5µl</td> <td>1.75</td> <td>5</td> </tr> | <tr align=right> <td>rg0302 100 µM</td> <td>0.5µl</td> <td>1.75</td> <td>5</td> </tr> | ||
<tr align=right> <td>Phusion</td> <td>1µl</td> <td>3.5</td> <td>10</td> </tr> | <tr align=right> <td>Phusion</td> <td>1µl</td> <td>3.5</td> <td>10</td> </tr> | ||
<tr><td></td></tr> | <tr><td></td></tr> | ||
<tr align=right> <td>template DNA</td> <td>0.1µl</td> <td>--</td> <td>--</td> </tr> | <tr align=right> <td>template DNA</td> <td>0.1µl</td> <td>--</td> <td>--</td> </tr> | ||
</table> | </table> | ||
Revision as of 07:34, 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
- Step 1: *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 | -- | -- |
- Step 2: *PCR program*
- 30"@98C;
- 35x (10"@98°C; 15"@68C; 1'30"@72C);
- 10'@72C;
- inf. 4C
- Keep at 4°C.
- Step 3
- Step 3 has multiple sub-steps within it.
- Enumerate each of those.
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.