Difference between revisions of "Team:British Columbia/Notebook/Protocols/Primers"

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  <h1>PCR Protocol using Phusion High-Fidelity DNA Polymerase</h1>
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<div class="UBCt"><div id="UBCt">
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<div id="one" style="width:400px;">&nbsp;</div>
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<div id="three" style="width:400px; margin-top:-25px;"><h3>A Primer on Primers</h3></div>
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    <div id="two" style="width:400px;">&nbsp;</div>
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<p><h2>Overview</h2>
 
<p><h2>Overview</h2>
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<p><strong>PCR&nbsp;</strong><br />
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<p><strong>Primers:</strong><br />
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<br />
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Phusion High-Fidelity DNA Polymerases, a DNA binding domain is fused to a Pyrococcus-like proofreading polymerase. Due to this unique fusion technique, Phusion DNA Polymerases generate PCR products with accuracy and speed unattainable with a single enzyme, even on the most difficult templates.</p>
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<h2>Protocol </h2>
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<p><strong>Reaction setup:</strong>&nbsp;<br />
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<br />
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Assemble all reaction components on ice and quickly transfer the reactions to a thermocycler preheated to the denaturation temperature (95&deg;C).&nbsp;<d>
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</d>
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<table class="table table-striped">
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      <thead>
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        <tr>
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          <th>Component</th>
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          <th>10 µl Reaction</th>
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          <th>50 µl Reaction</th>
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          <th>Final Concentration</th>
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        </tr>
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      </thead>
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      <tbody>
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        <tr>
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          <td>5X Phusion HF or GC Buffer</td>
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          <td>2 µl</td>
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          <td>10 µl </td>
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          <td>1X</td>
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        </tr>
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        <tr>
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          <td>10 mM dNTPs</td>
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          <td>0.2 µl</td>
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          <td>1 µl</td>
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          <td>200 µM</td>
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        </tr>
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        <tr>
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          <td>10 µM Forward Primer</td>
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          <td>0.5 µl</td>
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          <td>2.5 µl</td>
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          <td>0.5 µM</td>
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        </tr>
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        <tr>
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          <td>10 µM Reverse Primer</td>
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          <td>0.5 µl</td>
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          <td>2.5 µl</td>
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          <td>0.5 µM</td>
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        </tr>
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        <tr>
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          <td>Template DNA</td>
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          <td>Variable</td>
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          <td>Variable</td>
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          <td>< 250 ng</td>
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        </tr>
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        <tr>
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          <td>Phusion DNA Polymerase</td>
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          <td>0.1 µl</td>
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          <td>0.5 µl</td>
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          <td>1.0 units/50 µl PCR</td>
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        </tr>
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        <tr>
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          <td>Nuclease-free water</td>
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          <td>to 10 µl </td>
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          <td>to 50 µl</td>
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        </tr>
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      </tbody>
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    </table>
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<strong>Notes:</strong>
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<p>A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. Many of the laboratory techniques of molecular biology, such as DNA sequencing and the polymerase chain reaction (PCR), require DNA primers. These primers are usually short, chemically synthesized oligonucleotides, with a length of about twenty bases.</p>  
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<p>Before adding <em>Taq</em> DNA Polymerase, mix the reaction mixtures (or master mix) well. Once <em>Taq</em> DNA Polymerase has been added, gently mix by slowly pipetting up and down.</p> 
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Collect all liquid to the bottom of the tube by a quick spin if necessary.<br />
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<br />
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Transfer PCR tubes from ice to a PCR machine with&nbsp;the block preheated to 95&deg;C and begin thermocycling. <br />
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<br />
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<strong>Thermocycling conditions for a routine PCR using <em>Taq</em> DNA Polymerase</strong><br />
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<br />
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<em>This document will give you an overview on the proper methods of dealing with primers. What to do when they arrive, how to dilute them, and how to store them.</em>
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<h2>Protocol </h2>
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<strong><br />
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Dealing with newly arrived primers:</strong>&nbsp;</p>
  
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<p>Primers arrive in screw-top tubes, usually with a blue lid, containing freeze dried oligonucleotides. To use these primers, follow the steps below:</p>
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      <thead>
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          <th>Step</th>
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          <th>Temperature</th>
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          <th>Time</th>
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          <td>Initial Denaturation </td>
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          <td>98°C</td>
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          <td>30 seconds </td>
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          <td>
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  <div class="col-md-4">30 cycles:
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  <div class="col-md-4"><table>
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    <tr><td>Denaturation</td></tr>
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    <tr><td>Annealing</td></tr>
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    <tr><td>Extension</td></tr>
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    </table></div>
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</td>
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          <td>   
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<table>
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    <tr><td>98°C</td></tr>
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    <tr><td>45-72°C</td></tr>
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    <tr><td>72°C</td></tr>
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    </table>
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          <td>
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    <tr><td>15-30 seconds</td></tr>
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    <tr><td>15-60 seconds</td></tr>
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    <tr><td>15-30 seconds/kb</td></tr>
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</td>
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          <td>Final Extension</td>
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          <td>72°C</td>
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          <td>5 minutes</td>
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          <td>Hold</td>
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          <td>4°C</td>
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          <td>Infinite</td>
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<strong><br />
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General Guidelines:</strong>&nbsp;</p>
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<ol>
 
<ol>
 
     <li>
 
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     Template:&nbsp;<br />
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     Arrival:&nbsp;<br />
 
     <br />
 
     <br />
 
     Use of high quality, purified DNA templates greatly enhances the success of PCR:
 
     Use of high quality, purified DNA templates greatly enhances the success of PCR:
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          <th>DNA</th>
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          <th>Amount</th>
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          <td>Genomic</td>
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          <td>50 ng-250 ng</td>
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          <td>Plasmid</td>
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          <td>1 pg-10 ng</td>
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Latest revision as of 05:44, 22 August 2015

UBC iGEM 2015

 

A Primer on Primers

 

Overview

Primers:

A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. It is required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. Many of the laboratory techniques of molecular biology, such as DNA sequencing and the polymerase chain reaction (PCR), require DNA primers. These primers are usually short, chemically synthesized oligonucleotides, with a length of about twenty bases.

This document will give you an overview on the proper methods of dealing with primers. What to do when they arrive, how to dilute them, and how to store them.

Protocol


Dealing with newly arrived primers: 

Primers arrive in screw-top tubes, usually with a blue lid, containing freeze dried oligonucleotides. To use these primers, follow the steps below:

  1. Arrival: 

    Use of high quality, purified DNA templates greatly enhances the success of PCR:
  2. Primers: 

    Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 (http://frodo.wi.mit.edu/primer3) can be used to design or analyze primers. The final concentration of each primer in a reaction may be 0.05–1 μM, typically 0.1–0.5 μM.

  3. Mg++ and additives: 

    Mg++ concentration of 1.5–2.0 mM is optimal for most PCR products generated with Taq DNA Polymerase. The final Mg++ concentration in 1X Standard Taq Reaction Buffer is 1.5 mM. This supports satisfactory amplification of most amplicons. However, Mg++ can be further optimized in 0.5 or 1.0 mM increments using MgCl2. 

    Amplification of some difficult targets, like GC-rich sequences, may be improved with additives, such as DMSO (3) or formamide (4).

  4. Deoxynucleotides:

    The final concentration of dNTPs is typically 200 μM of each deoxynucleotide.

  5. Taq DNA Polymerase Concentration: 

    We generally recommend using Taq DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 μl reaction). However, the optimal concentration of Taq DNA Polymerase may range from 5–50 units/ml (0.25–2.5 units/50 μl reaction) in specialized applications.

  6. Denaturation: 

    An initial denaturation of 30 seconds at 95°C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, a longer initial denaturation of 2–4 minutes at 95°C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 95°C is recommended. 

    During thermocycling a 15–30 second denaturation at 95°C is recommended.

  7. Annealing: 

    The annealing step is typically 15–60 seconds. Annealing temperature is based on the Tm of the primer pair and is typically 45–68°C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5°C below the calculated Tm.  The NEB Tm Calculator is recommended to calculate an appropriate annealing temperature.

    When primers with annealing temperatures above 65°C are used, a 2-step PCR protocol is possible (see #10). 

  8. Extension: 

    The recommended extension temperature is 68°C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68°C is recommended.

  9. Cycle number: 

    Generally, 25-35 cycles yields sufficient product.