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Team:Goettingen/protocol PCR
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<h2 id="Pfus_PCR">Pfusion PCR</h2> | <h2 id="Pfus_PCR">Pfusion PCR</h2> | ||
| - | <p>Watch the following tutorial to learn the whole process:<br /><br /></p> | + | <p>Watch the following tutorial to learn about the whole process:<br /><br /></p> |
<iframe width="640" height="390" src="//www.youtube.com/embed/gT2yVxLlfCQ" frameborder="0" allowfullscreen></iframe> | <iframe width="640" height="390" src="//www.youtube.com/embed/gT2yVxLlfCQ" frameborder="0" allowfullscreen></iframe> | ||
| + | <br /><br /> | ||
<p>Reaction protocol for 50 µl<br /> 1 μl Primer fwd (5 pmol)<br /> 1 μl Primer rev (5 pmol)<br /> 1 μl Matrizen-DNA (ca. 100 ng)<br /> 10 μl 5x HF-Polymerase Puffer<br /> 0.5 μl PfuS-Polymerase (50 ng)<br /> 1 μl dNTPs (12.5 μmol ml-1)<br /> 35.5 μl dest. Water<br /><br /> | <p>Reaction protocol for 50 µl<br /> 1 μl Primer fwd (5 pmol)<br /> 1 μl Primer rev (5 pmol)<br /> 1 μl Matrizen-DNA (ca. 100 ng)<br /> 10 μl 5x HF-Polymerase Puffer<br /> 0.5 μl PfuS-Polymerase (50 ng)<br /> 1 μl dNTPs (12.5 μmol ml-1)<br /> 35.5 μl dest. Water<br /><br /> | ||
PCR cycles:</p> | PCR cycles:</p> | ||
Revision as of 13:36, 9 September 2014
Overview
PCR Methods
Plasmid Construction
- Restriction of DNA
- Ligation of DNA fragments
- BP recombination reaction
- LR recombination reaction
- SEAMLESS Cloning
- Peptide Library construction
Plasmid Transformation
- E.coli competent cells
- Plasmid isolation (E.coli)
- E.coil transformation
- Plasmid isolation (Yeast)
- Yeast transformation
Colony Scanning
Protein Assessment
In vivo tests
Pfusion PCR
Watch the following tutorial to learn about the whole process:
Reaction protocol for 50 µl
1 μl Primer fwd (5 pmol)
1 μl Primer rev (5 pmol)
1 μl Matrizen-DNA (ca. 100 ng)
10 μl 5x HF-Polymerase Puffer
0.5 μl PfuS-Polymerase (50 ng)
1 μl dNTPs (12.5 μmol ml-1)
35.5 μl dest. Water
PCR cycles:
| Cycles | Reaction | Temp. | Time |
| 1 | Pre-running | 98 °C | 10 min |
| 30 | Denature | 98 | 10 s |
| 30 | Annealing | 54-60 °C | 1 min |
| 30 | Primer Extension | 72 °C | 15 s - 2.5 min |
| 1 | Finish | 72 °C | 10 min |
