Team:Calgary/Notebook/Protocols/taqpcr

From 2012.igem.org

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<td width="85%"><div class="heading"><h3></a>Mastermix setup</h3> </p></div></td>
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<td width="85%"><div class="heading"><center><h3></a>Mastermix setup</h3> </center></p></div></td>
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   <p><i>Thermocycler Conditions</i></p>
   <p><i>Thermocycler Conditions</i></p>
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     <li> 1 Cycle - 6 minutes at 95&deg;C </li>
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     <li> 1 Cycle - 10 minutes at 95&deg;C </li>
     <li> 36 cycles of:
     <li> 36 cycles of:
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         <li type="a"> 1 minute at 95&deg;C </li>
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         <li type="a"> 3 minutes at 95&deg;C </li>
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         <li type="a"> 1 minute at 58&deg;C (this step done at 65&deg;C for higher GC content ) </li>
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         <li type="a"> 1 minute at 55&deg;C (for BioBrick primers) </li>
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         <li type="a"> 1 minute at 72&deg;C </li>
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         <li type="a"> 1 minute/kb at 72&deg;C </li>
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   <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>
<p>Follow the same protocol for a colony PCR.</p>
<p>Follow the same protocol for a colony PCR.</p>
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<p>We also used a Taq polymerase that we isolated in-house with slightly different cycling conditions. Since this Taq could not tolerate the lysis stage at 95&deg;C for 10 minutes, the cells were lysed in 4 μL of water for 10 minutes before master mix was added. 
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Latest revision as of 03:54, 4 October 2012

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Taq PCR Protocol

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

Mastermix setup

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 - 10 minutes at 95°C
  2. 36 cycles of:
    1. 3 minutes at 95°C
    2. 1 minute at 55°C (for BioBrick primers)
    3. 1 minute/kb 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.

We also used a Taq polymerase that we isolated in-house with slightly different cycling conditions. Since this Taq could not tolerate the lysis stage at 95°C for 10 minutes, the cells were lysed in 4 μL of water for 10 minutes before master mix was added.