Engineering BioBrick vectors from BioBrick parts/Colony PCR protocol

From OpenWetWare
Revision as of 23:29, 19 November 2009 by Vaishnavi Ananth (talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to: navigation, search

<html><h2>Solutions/reagents:</h2><ul type="circle"><li>PCR SuperMix High Fidelity</li><li> <a name="VF2 primer">VF2 primer <i><br><tab><div style="margin-right: 600px;">(5'-TGCCACCTGACGTCTAAGAA-3')</div></i></a></li><li> <a name="VR primer">VR primer <i><br><tab><div style="margin-right: 600px;">(5'-ATTACCGCCTTTGAGTGAGC-3')</div></i></a></li><li> <a name="colony suspension">colony suspension <i><br><tab><div style="margin-right: 600px;">(1 colony diluted in 100 ?l water)</div></i></a></li><li>de-ionized water</li></ul><h2>Parameters:</h2><ul type="circle"><li>X - concentration of primers (µmole/µl)</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Sterile 0.6-ml tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>PCR mix</font></b><br>Use the following table as a checklist for preparing the reaction in sterile 0.6-ml microcentrifuge tube (1):<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>PCR SuperMix High Fidelity</font></td><td><font color=#357EC7>VF2 primer</font></td><td><font color=#357EC7>VR primer</font></td><td><font color=#357EC7>colony suspension</font></td><td><font color=#357EC7>de-ionized water</font></td></tr></thead><tbody><tr><td><font color=#357EC7>Colony PCR</font></td><td><b><b><font color=#357EC7>9 µl</font></b></td><td><b><b><font color=#357EC7>0.125/X</font></b></td><td><b><b><font color=#357EC7>0.125/X</font></b></td><td><b><b><font color=#357EC7>1 µl</font></b></td><td><b><font color=#357EC7>Make up the volume to <b><font color=#357EC7>20 µl</font></b></font></b></td></tr></body></table></li></p><p><li><b><font size=3>PCR conditions</font></b><br>Program a standard thermocycler to run the reaction using the following parameters:<br>Initial denaturation<br><ul><li>Denature: <b><font color=#357EC7>95°C</font></b>, <b><font color=#357EC7>15 mins</font></b></li></ul>Thermocycling<br><ul><li>No. of cycles: <b><font color=#357EC7>39</font></b></li><li>Denature: <b><font color=#357EC7>94°C</font></b>, <b><font color=#357EC7>30 secs</font></b></li> <li> Anneal: <b><font color=#357EC7>62°C</font></b>, <b><font color=#357EC7>30 secs</font></b></li> <li>Elongate: <b><font color=#357EC7>68°C</font></b>, <b><font color=#357EC7>3.5 mins</font></b></li></ul>Termination<ul><li>Elongate: <b><font color=#357EC7>68°C</font></b>, <b><font color=#357EC7>20 mins</font></b></li><li>Hold: <b><font color=#357EC7>4°C</font></b>, until removed from machine </li></ul></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 3 hrs, 35 mins</font></b></p></html>