Team:Calgary/Notebook/Protocols/Process9

From 2011.igem.org

(Difference between revisions)
(Created page with "{{Team:Calgary/Main_Header|notebook}} {{Team:Calgary/Notebookbar| TITLE=Plasmid Extraction| BODY=<html> <div style="margin-bottom:60px"> <table width="700"> <tr> <t...")
 
(16 intermediate revisions not shown)
Line 2: Line 2:
{{Team:Calgary/Notebookbar|
{{Team:Calgary/Notebookbar|
-
TITLE=Plasmid Extraction|
+
TITLE=Taq PCR Protocol|
BODY=<html>
BODY=<html>
 +
 +
<p>The following is obtained from the Invitrogen Taq PCR kit (although most PCR kits have similar protocols).</p>
<div style="margin-bottom:60px">
<div style="margin-bottom:60px">
Line 9: Line 11:
     <tr>
     <tr>
       <td width="85%"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="pcr"></a>Taq PCR Protocol </p></div></td>
       <td width="85%"><div class="heading"><p style="font-size:14px; font-weight:bold"><a style="text-decoration:none" name="pcr"></a>Taq PCR Protocol </p></div></td>
-
      <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td>
 
     </tr>
     </tr>
   </table>
   </table>
Line 72: Line 73:
   <p><b> Thermocycler Conditions </b></p>
   <p><b> Thermocycler Conditions </b></p>
   <ol>
   <ol>
-
     <li> 1 Cycle - 6 minutes at 95 degrees Celsius </li>
+
     <li> 1 Cycle - 6 minutes at 95&deg;C </li>
     <li> 36 cycles of:
     <li> 36 cycles of:
       <ol>
       <ol>
-
         <li> 1 minute at 95 degrees Celsius </li>
+
         <li type="a"> 1 minute at 95&deg;C </li>
-
         <li> 1 minute at 58 degrees Celsius ( this step done at 65 degrees Celsius for higher GC content ) </li>
+
         <li type="a"> 1 minute at 58&deg;C (this step done at 65&deg;C for higher GC content ) </li>
-
         <li> 1 minute at 72 degrees Celsius </li>
+
         <li type="a"> 1 minute at 72&deg;C </li>
       </ol>
       </ol>
     </li>
     </li>
-
     <li> 1 Cycle - 10 minutes at 72 degrees Celsius then HOLD at 4 degrees Celsius </li>
+
     <li> 1 Cycle - 10 minutes at 72&deg;C then HOLD at 4&deg;C</li>
   </ol>
   </ol>
   <p> Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 or even 3 minutes</p>
   <p> Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 or even 3 minutes</p>
Line 87: Line 88:
</td>
</td>
</tr>
</tr>
-
 
+
<p>Follow the same protocol for a colony PCR.</p>
-
<tr>
+
</html>}}
-
<td width="750" bgcolor="#222222" height="10">
+
-
</td>
+
-
</tr>
+
-
 
+
-
<tr>
+
-
<td>
+

Latest revision as of 04:26, 29 September 2011


Taq PCR Protocol

The following is obtained from the Invitrogen Taq PCR kit (although most PCR kits have similar protocols).

Taq PCR Protocol

Reagent Volume ( 1x ) Volume ( 3x ) Volume ( 5x ) Volume ( 15x )
Sterile H2O 36 μL 108 μL 180 μL 540 μL
10X Taq Buffer 5 μL 15 μL 25 μL 75 μL
2mM dNTPs 5 μL 15 μL 25 μL 75 μL
Forward Primer (100 ug/ul) 1 μL 3 μL 5 μL 15 μL
Reverse Primer (100 ug/ul) 1 μL 3 μL 5 μL 15 μL
50mM MgCl2 1.5 μL 4.5 μL 7.5 μL 22.5 μL
Taq Polymerase (50 ug/ul) 0.5 μL 1.5 μL 2.5 μL 7.5 μL

Thermocycler Conditions

  1. 1 Cycle - 6 minutes at 95°C
  2. 36 cycles of:
    1. 1 minute at 95°C
    2. 1 minute at 58°C (this step done at 65°C for higher GC content )
    3. 1 minute at 72°C
  3. 1 Cycle - 10 minutes at 72°C then HOLD at 4°C

Conditions were varied as needed. For example in cases of longer products all 1 minute times were increased to 1.5 or even 3 minutes


Follow the same protocol for a colony PCR.

Retrieved from "http://2011.igem.org/Team:Calgary/Notebook/Protocols/Process9"