Team:BYU Provo/Notebook/CommonProcedures

From 2014.igem.org

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<h><blockquote><b>Q5 PCR Reaction:</b></blockquote></h>
 
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<p><blockquote>10 uL 5x Q5 Reaction Buffer</p></blockquote>
 
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<p><blockquote>1 uL dNTPs</p></blockquote>
 
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<p><blockquote>1 uL Forward Primer</p></blockquote>
 
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<p><blockquote>1 uL Reverse Primer</p></blockquote>
 
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<p><blockquote>10 uL Q5 Enhancer</p></blockquote>
 
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<p><blockquote>23.5 uL ddH2O</p></blockquote>
 
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<p><blockquote>1-2 uL Template</p></blockquote>
 
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<p><blockquote>---------------</p></blockquote>
 
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<p><blockquote>50 uL Reaction</blockquote></p>
 
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<br></br>
 
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<p><blockquote> uL Q5 Polymerase (add right before you start the PCR reaction. Keep on ice until you are ready to do so.)</blockquote></p>
 
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<h2>TAQ PCR (50 uL Reaction)</h2>
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<br></br>
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<h2>For TAQ PCR (50 uL reaction)</h2>
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<ul><li>35 ul ddH20</li>
<ul><li>35 ul ddH20</li>
<li>10  ul 5X REDTAQ buffer (mix well before use!!)</li>
<li>10  ul 5X REDTAQ buffer (mix well before use!!)</li>
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<li>1 ul appropriate diluted template DNA </li>
<li>1 ul appropriate diluted template DNA </li>
<li>Mix well then add 2.5 ul REDTAQ polymerase</li></ul>
<li>Mix well then add 2.5 ul REDTAQ polymerase</li></ul>
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<p>Set up reactions on ice, and keep them on ice until placing them on the PCR machine which has been pre-warmed to 94°C (this is called a “hot start”). Abundant templates only require 20-25 cycles for amplification; dilute/complex templates require 35-40 cycles. Extension times vary depending on target size.</p>
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<p>Set up reactions on ice, and keep them on ice until placing them on the PCR machine which has been pre-warmed to 94°C. Abundant templates only require 20-25 cycles for amplification; dilute/complex templates require 35-40 cycles. Extension times vary depending on target size.</p>
 +
 
 +
<h2>Q5 PCR Reaction (50 uL Reaction)</h2>
 +
<ul><li>10 uL 5x Q5 Reaction Buffer</li>
 +
<li>1 uL dNTPs</li>
 +
<li>1 uL Forward Primer</li>
 +
<li>1 uL Reverse Primer</li>
 +
<li>10 uL Q5 Enhancer</li>
 +
<li>23.5 uL ddH2O</li>
 +
<li>1-2 uL Template</li>
 +
<li>Add ?uL Q5 Polymerase right before you start the PCR reaction.</li></ul>
 +
<p>Set up reactions on ice, making sure to keep Q5 Polymerase on ice until needed.</p>
 +
 
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<h2>Phusion PCR</h2>
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<ul><li>35 ul ddH20</li>
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<li>10  ul 5X Phusion GC buffer</li>
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<li>1.0 uL DMSO</li>
 +
<li>1.5 ul 10 mM dNTP’s</li>
 +
<li>1 ul each Primer (50 uM stock)</li>
 +
<li>1 ul appropriate diluted template DNA</li>
 +
<li>0.5 ul Phusion Polymerase</li></ul>
 +
 
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Revision as of 00:53, 22 July 2014


BYU 2014 Notebook

EDIT Procedures

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TAQ PCR (50 uL Reaction)

  • 35 ul ddH20
  • 10 ul 5X REDTAQ buffer (mix well before use!!)
  • 1.0 ul 10 mM dNTP’s
  • 1 ul each Primer (50 uM stock)
  • 1 ul appropriate diluted template DNA
  • Mix well then add 2.5 ul REDTAQ polymerase

Set up reactions on ice, and keep them on ice until placing them on the PCR machine which has been pre-warmed to 94°C. Abundant templates only require 20-25 cycles for amplification; dilute/complex templates require 35-40 cycles. Extension times vary depending on target size.

Q5 PCR Reaction (50 uL Reaction)

  • 10 uL 5x Q5 Reaction Buffer
  • 1 uL dNTPs
  • 1 uL Forward Primer
  • 1 uL Reverse Primer
  • 10 uL Q5 Enhancer
  • 23.5 uL ddH2O
  • 1-2 uL Template
  • Add ?uL Q5 Polymerase right before you start the PCR reaction.

Set up reactions on ice, making sure to keep Q5 Polymerase on ice until needed.

Phusion PCR

  • 35 ul ddH20
  • 10 ul 5X Phusion GC buffer
  • 1.0 uL DMSO
  • 1.5 ul 10 mM dNTP’s
  • 1 ul each Primer (50 uM stock)
  • 1 ul appropriate diluted template DNA
  • 0.5 ul Phusion Polymerase

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