Knight:Colony PCR protocol

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(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li>PCR supermix</li><li> <a name="VF2">VF2 <i><br><tab><div style="margin-right: 600px;">(40 µM)</div></i></a></li><li> <a name="VR">V...)
Current revision (02:27, 20 November 2009) (view source)
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>PCR supermix</li><li> <a name="VF2">VF2 <i><br><tab><div style="margin-right: 600px;">(40 µM)</div></i></a></li><li> <a name="VR">VR <i><br><tab><div style="margin-right: 600px;">(40 µM)</div></i></a></li><li>colony template</li></ul><h2>Parameters:</h2><ul type="circle"><li>X - 1 min per kb of expected product.</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Electrophoretic unit</li><li>Reaction tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Reaction Mixture</font></b><br>Use the following table as a checklist for preparing the reaction:<br><br><table border cellpadding=5 rules=all frame=void bordercolor=#357EC7><thead><tr><td>&nbsp;</td><td><font color=#357EC7>PCR supermix</font></td><td><font color=#357EC7>VF2</font></td><td><font color=#357EC7>VR</font></td><td><font color=#357EC7>colony template</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>0.25 µl</font></b></td><td><b><b><font color=#357EC7>0.25 µl</font></b></td><td><b><b><font color=#357EC7>0.5 µl</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>56°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>X</font></b></li></ul><font color = "#800517"><i>Elongation time : I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.</i></font><br>Termination<ul><li>Elongate: <b><font color=#357EC7>60°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><p><li>Perform agarose gel electrophoresis of appropriate quantity of  PCR products mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.<br></li></p></ol></html>
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>PCR supermix</li><li> <a name="VF2">VF2 <i><br><tab><div style="margin-right: 600px;">(40 µM)</div></i></a></li><li> <a name="VR">VR <i><br><tab><div style="margin-right: 600px;">(40 µM)</div></i></a></li><li>colony template</li></ul><h2>Equipment:</h2><ul type="circle"><li>Thermocycler</li><li>Electrophoretic unit</li><li>Reaction tubes</li></ul><h2>Steps:</h2><ol><p><li><b><font size=3>Reaction Mixture</font></b><br>Use the following table as a checklist for preparing the reaction in reaction 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</font></td><td><font color=#357EC7>VF2</font></td><td><font color=#357EC7>VR</font></td><td><font color=#357EC7>colony template</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>0.25 µl</font></b></td><td><b><b><font color=#357EC7>0.25 µl</font></b></td><td><b><b><font color=#357EC7>0.5 µl</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>56°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>60 secs</font></b></li></ul><font color = "#800517"><i>Elongation time : 1 min per kb of expected product. I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.</i></font><br>Termination<ul><li>Elongate: <b><font color=#357EC7>60°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><p><li>Perform agarose gel electrophoresis of appropriate quantity of  PCR products mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 1 hr, 55 mins</font></b></p></html>

Current revision

Solutions/reagents:

Equipment:

  • Thermocycler
  • Electrophoretic unit
  • Reaction tubes

Steps:

  1. Reaction Mixture
    Use the following table as a checklist for preparing the reaction in reaction tube (1):

     PCR supermixVF2VRcolony template
    Colony PCR9 µl0.25 µl0.25 µl0.5 µl
  2. PCR conditions
    Program a standard thermocycler to run the reaction using the following parameters:
    Initial denaturation
    • Denature: 95°C, 15 mins
    Thermocycling
    • No. of cycles: 39
    • Denature: 94°C, 30 secs
    • Anneal: 56°C, 30 secs
    • Elongate: 68°C, 60 secs
    Elongation time : 1 min per kb of expected product. I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.
    Termination
    • Elongate: 60°C, 20 mins
    • Hold: 4°C, until removed from machine
  3. Perform agarose gel electrophoresis of appropriate quantity of PCR products mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 55 mins

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