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Team:Hannover/Protocols/Cloning/PCR
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<h1><a href="https://2014.igem.org/Team:Hannover/Protocols" target="_blank">Protocols</a> / Standard and Colony PCR</h1> | <h1><a href="https://2014.igem.org/Team:Hannover/Protocols" target="_blank">Protocols</a> / Standard and Colony PCR</h1> | ||
| - | <p class="text">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 (Table 1)are summarized Table 1 and 2 respectively. 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 | + | <p class="text">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 (Table 1)are summarized Table 1 and 2 respectively. 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 H<sub>2</sub>O first and then lysed by denaturation for 10 min at 80 °C. Before usage, the boiled bacteria solution was centrifuged for a short time. </p> |
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<h2>Table 1: Reaction Mixes and Temperature Programs for colony PCR.</h2> | <h2>Table 1: Reaction Mixes and Temperature Programs for colony PCR.</h2> | ||
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<tr><td><b>Volume [μl]</b></td><td><b>Compounds of standard PCR</b></td></tr> | <tr><td><b>Volume [μl]</b></td><td><b>Compounds of standard PCR</b></td></tr> | ||
<tr><td>4.00</td><td>5 x Green GoTaq© Reaction buffer</td></tr> | <tr><td>4.00</td><td>5 x Green GoTaq© Reaction buffer</td></tr> | ||
| - | <tr><td>1.00</td><td>25 mM µl-1 | + | <tr><td>1.00</td><td>25 mM µl-1 MgCl<sub>2</sub></td></tr> |
<tr><td>1.00</td><td>2.5 mM µl-1 dNTPs</td></tr> | <tr><td>1.00</td><td>2.5 mM µl-1 dNTPs</td></tr> | ||
<tr><td>0.25</td><td>10 mM µl-1 sense oligonucleotide</td></tr> | <tr><td>0.25</td><td>10 mM µl-1 sense oligonucleotide</td></tr> | ||
<tr><td>0.25</td><td>10 mM µl-1 reverse oligonucleotide</td></tr> | <tr><td>0.25</td><td>10 mM µl-1 reverse oligonucleotide</td></tr> | ||
<tr><td>0.30</td><td>GoTaq® DNA Polymerase</td></tr> | <tr><td>0.30</td><td>GoTaq® DNA Polymerase</td></tr> | ||
| - | <tr><td>0.50</td><td>10 – 100 ng template</td></tr><tr><td colspan="2">ad 20 µl | + | <tr><td>0.50</td><td>10 – 100 ng template</td></tr><tr><td colspan="2">ad 20 µl H<sub>2</sub>O</td></tr></table></td><td><table colspan="2" ><tr><td><b>Cycler Program</b></td></tr><tr><td>Step</td><td>Temperature [°C]</td><td>Time [min]</td><td>Cycle no.</td></tr><tr><td>1.</td><td>95</td><td>2.0</td><td>1</td></tr><tr><td>2.</td><td>95</td><td>0.5</td><td>35</td></tr><tr><td>3.</td><td>TA</td><td>0.5</td><td>35</td></tr><tr><td>4.</td><td>72</td><td>-</td><td>35</td></tr></table></td></tr></table> |
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<h2>Table 2: Reaction Mixes and Temperature Programs for standard PCR.</h2> | <h2>Table 2: Reaction Mixes and Temperature Programs for standard PCR.</h2> | ||
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<tr><td>0.25</td><td>10 mM µl-1 reverse oligonucleotide</td></tr> | <tr><td>0.25</td><td>10 mM µl-1 reverse oligonucleotide</td></tr> | ||
<tr><td>0.30</td><td>Phusion® High-Fidelity DNA Polymerase </td></tr> | <tr><td>0.30</td><td>Phusion® High-Fidelity DNA Polymerase </td></tr> | ||
| - | <tr><td>0.50</td><td>10 – 100 ng template</td></tr><tr><td colspan="2">ad 20 µl | + | <tr><td>0.50</td><td>10 – 100 ng template</td></tr><tr><td colspan="2">ad 20 µl H<sub>2</sub>O</td></tr></table></td><td><table colspan="2" ><tr><td><b>Cycler Program</b></td></tr><tr><td>Step</td><td>Temperature [°C]</td><td>Time [min]</td><td>Cycle no.</td></tr><tr><td>1.</td><td>98</td><td>2.0</td><td>1</td></tr><tr><td>2.</td><td>98</td><td>0.5</td><td>35</td></tr><tr><td>3.</td><td>TA</td><td>0.5</td><td>35</td></tr><tr><td>4.</td><td>72</td><td>-</td><td>35</td></tr></table></td></tr></table> |
<p id="Fusszeile"><br><br><br><br><img src="https://static.igem.org/mediawiki/2014/6/6a/Hannover_20141010Fusszeile2.jpg" width="980px" style="float:right" /></p> | <p id="Fusszeile"><br><br><br><br><img src="https://static.igem.org/mediawiki/2014/6/6a/Hannover_20141010Fusszeile2.jpg" width="980px" style="float:right" /></p> | ||
</body> | </body> | ||
</html> | </html> | ||
Revision as of 18:03, 17 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 (Table 1)are summarized Table 1 and 2 respectively. 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 colony PCR.
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Table 2: Reaction Mixes and Temperature Programs for standard PCR.
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