Difference between revisions of "Team:Northwestern/PCR"

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   <article>
 
   <article>
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<h1>PCR</h1>
 
<h1>PCR</h1>
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       <li>95&#176;C (5 min) initial denaturation</li>
 
       <li>95&#176;C (5 min) initial denaturation</li>
 
       <li>~25 cycles of:</li>
 
       <li>~25 cycles of:</li>
−
       <ul><li>50—70&#176;C (7–15 s) annealing</li>
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       <ul>
−
      <ul><li>Annealing temperature depends on the primers being used</li></ul>
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        <li>95&#176;C (7 s) denaturing</li>
−
      <li>72&#176;C (30—60 s per kb) elongation</li>
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        <li>50—70&#176;C (7–15 s) annealing</li>
−
      <ul><li>Elongation time depends on the length of the sequence being amplified</li></ul></ul>
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        <ul>
 +
          <li>Annealing temperature depends on the primers being used</li>
 +
        </ul>
 +
        <li>72&#176;C (30—60 s per kb) elongation</li>
 +
        <ul>
 +
          <li>Elongation time depends on the length of the sequence being amplified</li>
 +
        </ul>
 +
      </ul>
 
       <li>72&#176;C (5 min) final incubation</li>
 
       <li>72&#176;C (5 min) final incubation</li>
 +
      <li>* <strong>Touchdown PCR:</strong> Start 10&#176;C above annealing temperature and decrease the T<sub>a</sub> over 10 cycles to the final primer T<sub>a</sub> if primers are binding to alternate sites and giving incorrect products</li>
 
     </ul>
 
     </ul>
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    <li><strong>Touchdown PCR:</strong> Start 10&#176;C above annealing temperature and decrease the T<sub>a</sub> over 10 cycles to the final primer T<sub>a</sub> if primers are binding to alternate sites and giving incorrect products</li>
 
 
     <h2>Recipe (25 uL reaction):</h2>
 
     <h2>Recipe (25 uL reaction):</h2>
 
     <ul>
 
     <ul>
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       <li>0.25 µL polymerase</li>
 
       <li>0.25 µL polymerase</li>
 
       <li>Nuclease-free water to 25 uL</li>
 
       <li>Nuclease-free water to 25 uL</li>
−
      </ul>
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    </ul>
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     <h2>Using Master Mix (25 uL reaction):</h2>
−
      <li>Running with Master Mix</li>
+
    <ul>
−
      <ul><li>0.5 uL forward primer (10 uM)</li>
+
      <li>0.5 uL forward primer (10 uM)</li>
 
       <li>0.5 uL reverse primer (10 uM)</li>
 
       <li>0.5 uL reverse primer (10 uM)</li>
−
      <li>1 uL DMSO </li>
 
 
       <li>1–5 ng template DNA (less works better)</li>
 
       <li>1–5 ng template DNA (less works better)</li>
 +
      <li>1 uL DMSO </li>
 
       <li>12.5 uL Master Mix</li>
 
       <li>12.5 uL Master Mix</li>
−
       <li>Nuclease-free water to 25 uL </li></ul>
+
       <li>Nuclease-free water to 25 uL </li>
−
      <li>Run 50 uL reactions to have more product</li>
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    </ul>
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 +
 
  
 
</article>
 
</article>

Latest revision as of 20:58, 16 October 2016

Protocols

PCR

General Conditions:

  • 95°C (5 min) initial denaturation
  • ~25 cycles of:
    • 95°C (7 s) denaturing
    • 50—70°C (7–15 s) annealing
      • Annealing temperature depends on the primers being used
    • 72°C (30—60 s per kb) elongation
      • Elongation time depends on the length of the sequence being amplified
  • 72°C (5 min) final incubation
  • * Touchdown PCR: Start 10°C above annealing temperature and decrease the Ta over 10 cycles to the final primer Ta if primers are binding to alternate sites and giving incorrect products

Recipe (25 uL reaction):

  • 2.5 µL 10x buffer
  • 0.5 µL 10mM dNTPs
  • 0.5 µL forward primer (10 uM)
  • 0.5 uL reverse primer (10 uM)
  • 1–5 ng template DNA (less works better)
  • 1 uL DMSO
  • 0.25 µL polymerase
  • Nuclease-free water to 25 uL

Using Master Mix (25 uL reaction):

  • 0.5 uL forward primer (10 uM)
  • 0.5 uL reverse primer (10 uM)
  • 1–5 ng template DNA (less works better)
  • 1 uL DMSO
  • 12.5 uL Master Mix
  • Nuclease-free water to 25 uL