Team:Hannover/Protocols/Cloning/PCR

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<table border="0" class="spadtable">
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<tr><th>Volume [μl]</th><th>Compounds of standard PCR</th></tr>
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<tr><td>Volume [μl]</td><td>Compounds of standard PCR</td></tr>
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<tr><td>4.00</td><td>5 x Phusion® HF buffer</td></tr>
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<tr><td>1.00</td><td>2.5 mM µl-1 dNTPs</td></tr>
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<tr><td>0.25</td><td>10 mM µl-1  sense oligonucleotide</td></tr>
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<tr><td>0.25</td><td>10 mM µl-1  reverse oligonucleotide</td></tr>
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<tr><td>0.30</td><td>Phusion® High-Fidelity DNA Polymerase </td></tr>
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<tr><td>0.50</td><td>10 – 100 ng template</td></tr>
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<tr colspan="2"><td>ad 20 µl H2O</td></tr></table>
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<h2>Table 2: Reaction mixes and temperature programs for colony PCR.</h2>
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<tr><td><h4>Material:</h4>
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<tr><td>Centrifuge</td></tr><tr><td>100 mM CaCl2</td></tr>
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<tr><td>86 % glycerin</td></tr></table><br></br>
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<p><h4>Protocol:</h4></p>
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<h2>Transformation of <i>E. coli</i> cells</h2>
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<table colspan="2"><tr><td><h4>Material:</h4></td>
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<tr><td>watherbath</td><td></td></tr><tr><td>shacking and stationary incubator</td><td></td></tr><tr><td>antibiotics</td><td></td></tr><tr><td></td><td></td></tr><tr><td>SOC-medium</td><td>937.5 μl SOB media,<br>12.5 μl 2 M MgCl2,<br>50.0 μl  20 % (w/v) glucose</td></tr><tr><td></td><td></td></tr><tr><td>SOB-medium</td><td>2.00 % (w/v) Tryptone,<br>0.50 % (w/v) Yeast extract,<br>0.06 % 10 mM NaCl,<br>0.02 % 2,5 mM KCl</td></tr><tr><td></td><td></td></tr><tr><td></td><td></td></tr><tr><td>2TY media</td><td>1.6 % (w/v) Peptone,<br>1.0 % (w/v) Yeast extract,<br>0.5 % (w/v) NaCl ; pH 7</td></tr></table>
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<p><h4>Protocol:</h4></p>
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<p class="text">Approximately 3-20 μl of ligation product was pipetted into 50 μl of freshly prepared heatshock competent <i>E. coli</i> cells. The mixture stayed on ice for 20 min, before the heatshock was conducted for 45 s at 42 °C. Afterwards 500 μl SOC media was added and the transformed bacteria were cultivated for 45 min shaking with 180 rpm at 37&nbsp;°C. Subsequently, the bacteria were grown on top of solid, antibiotics containing 2TY media. To allow optimal colony growth, the medium was stored at 37 °C overnight.</p>
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Revision as of 01:05, 14 October 2014

Protocols / Standard and Colony PCR

DNA amplification for important cloning or sequencing steps was performed by a standard PCR mix including the F-530 Phusion® High-Fidelity DNA Polymerase (Thermo Scientific). Due to its 3´-5´ exonuclease activity, it is capable of proofreading its product and thus works more accurately than the GoTaq® DNA Polymerase. Hence, the latter was used only for experiments in which a correct sequence had no importance, e.g. colony PCRs. Composition and temperature program for both, the colony and standard PCR are summarized in the following table. Contrary to the standard PCR, the nucleic acid template for colony PCRs was directly obtained by bacterial clones. To use them as templates, bacteria were transferred into 20 μl of H2O first and then lysed by denaturation for 10 min at 80 °C. Before usage, the boiled bacteria solution was centrifuged for a short time.

Table 1: Reaction mixes and temperature programs for standard PCR.

Volume [μl]Compounds of standard PCR
4.005 x Phusion® HF buffer
1.002.5 mM µl-1 dNTPs
0.2510 mM µl-1 sense oligonucleotide
0.2510 mM µl-1 reverse oligonucleotide
0.30Phusion® High-Fidelity DNA Polymerase
0.5010 – 100 ng template
ad 20 µl H2O

Table 2: Reaction mixes and temperature programs for colony PCR.





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