Difference between revisions of "Team:Freiburg/Protocols/PCR"
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− | <h3 class="sectionedit2"><a name="programm_normale_pcr" id="programm_normale_pcr"> | + | |
+ | <p> | ||
+ | |||
+ | <h3 class="sectionedit2"><a name="programm_normale_pcr" id="programm_normale_pcr">Standard PCR Programm</a></h3> | ||
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+ | <p> | ||
+ | For amplification of different DNA fragments from plasmid templates, various PCR approaches were used. In general, PCRs were performed with Phusion High Fidelity DNA Polymerase (NEB). The components of the reaction mixture were used in defined amounts/concentrations recommended by the manufacturer. | ||
+ | </p> | ||
+ | <p> | ||
+ | <body> | ||
+ | <h3>PCR mix</h3> | ||
+ | <table class="tabelle"> | ||
+ | <tr><th><center>ingredient</center></th><th><center>volume</center></th></tr> | ||
+ | <tr><td><center>Template</center></td><td><center>200 - 300 ng</center></td></tr> | ||
+ | <tr><td><center>Primer (10 µM)</center></td><td><center>1 µl each</center></td></tr> | ||
+ | <tr><td><center>Q5 Polymerase</center></td><td><center>0.5 µl</center></td></tr> | ||
+ | <tr><td><center>dNTPs</center></td><td><center>4 µl</center></td></tr> | ||
+ | <tr><td><center>DMSO</center></td><td><center>1 µl</center></td></tr> | ||
+ | <tr><td><center>Q5 reaction buffer (5x)</center></td><td><center>10 µl</center></td></tr> | ||
+ | <tr><td><center>dH<sub>2</sub>O</center></td><td><center>up to 50 µl</center></td></tr> | ||
+ | </table> | ||
+ | Add GC enhancer if the GC content of the template [??] is very high. | ||
+ | </body> | ||
+ | </p> | ||
+ | |||
+ | <p>Primers for DNA amplification were designed to have a melting temperature of about 60°C (detailed information can be found in the Oligo list). The following cycling conditions have been used for standard PCRs. Extention times were calculated according to NEB's recommendations for Phusion High-Fidelity Polymerase. | ||
+ | </p> | ||
<p> | <p> | ||
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− | <td class="col0 leftalign"> 98°C </td><td class="col1 leftalign"> 5 min </td><td class="col2 leftalign"> | + | <td class="col0 leftalign"> 98°C </td><td class="col1 leftalign"> 5 min </td><td class="col2 leftalign"> Initial denaturation </td> |
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− | <th class="col0 leftalign"> 98°C </th><th class="col1 leftalign"> 30 s </th><th class="col2 leftalign"> | + | <th class="col0 leftalign"> 98°C </th><th class="col1 leftalign"> 30 s </th><th class="col2 leftalign"> Denaturation </th> |
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− | <th class="col0 leftalign"> 72°C </th><th class="col1 leftalign"> 30 - 40 s pro kb </th><th class="col2 leftalign"> | + | <th class="col0 leftalign"> 72°C </th><th class="col1 leftalign"> 30 - 40 s pro kb </th><th class="col2 leftalign"> Extention </th> |
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− | <td class="col0 leftalign"> 72°C </td><td class="col1 leftalign"> 10 min </td><td class="col2 leftalign"> | + | <td class="col0 leftalign"> 72°C </td><td class="col1 leftalign"> 10 min </td><td class="col2 leftalign"> Final extention </td> |
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<a href="/igem2015/doku.php?id=tag:protocols_old&do=showtag&tag=protocols_old" class="wikilink1" title="tag:protocols_old" rel="tag">protocols old</a> | <a href="/igem2015/doku.php?id=tag:protocols_old&do=showtag&tag=protocols_old" class="wikilink1" title="tag:protocols_old" rel="tag">protocols old</a> |
Revision as of 14:47, 16 September 2015
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PCR
Standard PCR Programm
For amplification of different DNA fragments from plasmid templates, various PCR approaches were used. In general, PCRs were performed with Phusion High Fidelity DNA Polymerase (NEB). The components of the reaction mixture were used in defined amounts/concentrations recommended by the manufacturer.
PCR mix
Primers for DNA amplification were designed to have a melting temperature of about 60°C (detailed information can be found in the Oligo list). The following cycling conditions have been used for standard PCRs. Extention times were calculated according to NEB's recommendations for Phusion High-Fidelity Polymerase.
Lid temperature: 98°C
98°C | 5 min | Initial denaturation |
98°C | 30 s | Denaturation |
---|---|---|
63°C | 30 s | Annealing |
72°C | 30 - 40 s pro kb | Extention |
72°C | 10 min | Final extention |
4°C | hold |
18 - 25 Zyklen (Schritt 2-4)