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Team:Goettingen/protocol PCR
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<h3 id="Pfus_PCR"><i>PfuS</i> PCR</h3> | <h3 id="Pfus_PCR"><i>PfuS</i> PCR</h3> | ||
| - | <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 <i>PfuS</i>-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 /> |
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| + | 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 <i>PfuS</i>-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> | ||
<table class="table2" width="450" align="center"> | <table class="table2" width="450" align="center"> | ||
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<br /><br /> | <br /><br /> | ||
<p>Watch the following tutorial to learn about the whole process:<br /></p> | <p>Watch the following tutorial to learn about the whole process:<br /></p> | ||
| - | <center><video width="640" height="390 | + | <center><video width="640" height="390" controls><source src="https://static.igem.org/mediawiki/2014/7/7b/Goettingen2014-PCR.mp4" type="video/mp4">Your browser does not support the video tag.</video></center> |
<br /> | <br /> | ||
<p><a href="//www.youtube.com/watch?v=gT2yVxLlfCQ" target="_blank">HERE</a> you can find the high quality video.</p> | <p><a href="//www.youtube.com/watch?v=gT2yVxLlfCQ" target="_blank">HERE</a> you can find the high quality video.</p> | ||
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<h3 id="Fusi_PCR">Fusion PCR</h3> | <h3 id="Fusi_PCR">Fusion PCR</h3> | ||
<p>Reaction protocol 50 µl:<br /> | <p>Reaction protocol 50 µl:<br /> | ||
| - | + | <table class="table2"> | |
| - | + | <tr><td>4 μl</td><td>Primer forward (5 pmol)</td></tr> | |
| - | + | <tr><td>4 μl</td><td>Primer reverse (5 pmol)</td></tr> | |
| - | + | <tr><td>100 μl</td><td>template-DNA of each fragment</td></tr> | |
| - | + | <tr><td>10 μl</td><td>5x HF-Buffer</td></tr> | |
| - | + | <tr><td>1 μl</td><td><i>PfuS</i>-Polymerase</td></tr> | |
| - | + | <tr><td>2 μl</td><td>dNTPs (12.5 mM)</td></tr> | |
| - | + | <tr><td>add 50 μl</td><td>sterile water</td></tr> | |
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| + | </table> | ||
| + | |||
<table class="table2" align="center"> | <table class="table2" align="center"> | ||
<tr><th>Amount of cycles</th><th>Reaction </th><th> Temp.</th><th> Time</th></tr> | <tr><th>Amount of cycles</th><th>Reaction </th><th> Temp.</th><th> Time</th></tr> | ||
Latest revision as of 21:52, 17 October 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
PfuS PCR
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 | Final elongation | 72 °C | 10 min |
| 1 | Hold | 4 °C | ∞ |
Watch the following tutorial to learn about the whole process:
HERE you can find the high quality video.
Fusion PCR
Reaction protocol 50 µl:
| 4 μl | Primer forward (5 pmol) |
| 4 μl | Primer reverse (5 pmol) |
| 100 μl | template-DNA of each fragment |
| 10 μl | 5x HF-Buffer |
| 1 μl | PfuS-Polymerase |
| 2 μl | dNTPs (12.5 mM) |
| add 50 μl | sterile water |
| Amount of cycles | Reaction | Temp. | Time |
|---|---|---|---|
| 1 | Pre-running | 98°C | 1 min |
| 10 | Denaturation | 98°C | 15 sec |
| 10 | Annealing | 52°C | 30 sec |
| 10 | Primer Extension | 72°C | 2 min |
| 1 | Pause | 8°C | add primer |
| 21 | Denaturation | 98°C | 15 sec |
| 21 | Annealing | 52°C | 30 sec |
| 21 | Primer Extension | 72°C | 2 min + 5 sec/cycle |
| 1 | Final elongation | 72°C | 10 min |
| 1 | Hold | 4°C | ∞ |
E.coli colony PCR
1. Pick a fresh colony of about 1 mm and resuspend it in 10 µl water.
2. Use 1 µl as a template for a 25 µl PCR reaction.
Yeast colony PCR
1. Aliquot 20 µl NaOH (20 mM) into 1.5 ml tubes
2. Pick colonies (use pipet tips) into the NaOH
3. Incubate at 95°C for ~ 45 minutes
4. Centrifuge at max speed for 10 minutes
5. Use 5 µl of supernatant as template in a (50 µl) PCR.
