TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is for PCR with&nbsp;Q5&reg; High-Fidelity 2X Master Mix (M0492)

[GUIDELINES]
<strong>Please note that protocols with&nbsp;Q5 High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.</strong><br /><br /><strong>Reaction Setup:</strong><br />We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98&deg;C). All components should be mixed prior to use.<br />
<table cellspacing="0" cellpadding="1">
<tbody>
<tr><th><strong>Component</strong></th><th><strong>25 &micro;l Reaction</strong></th><th><strong>50 &micro;l Reaction</strong></th><th><strong>Final Concentration</strong></th></tr>
<tr>
<td>Q5&nbsp;High-Fidelity 2X Master Mix</td>
<td>12.5 &micro;l</td>
<td>25 &micro;l</td>
<td>1X</td>
</tr>
<tr>
<td>10 &micro;M Forward Primer</td>
<td>1.25 &micro;l</td>
<td>2.5 &micro;l</td>
<td>0.5 &micro;M</td>
</tr>
<tr>
<td>10 &micro;M Reverse Primer</td>
<td>1.25 &micro;l</td>
<td>2.5 &micro;l</td>
<td>0.5 &micro;M</td>
</tr>
<tr>
<td>Template DNA</td>
<td>variable</td>
<td>variable</td>
<td>&lt; 1,000 ng</td>
</tr>
<tr>
<td>Nuclease-Free Water</td>
<td>to 25 &micro;l</td>
<td>to 50 &micro;l</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.&nbsp;<br /><br />Transfer PCR tubes to a PCR machine and begin thermocycling.&nbsp;<br /><br /><strong>Thermocycling Conditions for a Routine PCR:</strong>
<table cellspacing="0" cellpadding="1">
<tbody>
<tr><th>STEP</th><th>TEMP</th><th>TIME</th></tr>
<tr>
<td>Initial Denaturation</td>
<td>98&deg;C</td>
<td>30 seconds</td>
</tr>
<tr>
<td rowspan="3">25&ndash;35 Cycles</td>
<td>98&deg;C</td>
<td>5&ndash;10 seconds</td>
</tr>
<tr>
<td>*50&ndash;72&deg;C</td>
<td>10&ndash;30 seconds</td>
</tr>
<tr>
<td>72&deg;C</td>
<td>20&ndash;30 seconds/kb</td>
</tr>
<tr>
<td>Final Extension</td>
<td>72&deg;C</td>
<td>2 minutes</td>
</tr>
<tr>
<td>Hold</td>
<td>4&ndash;10&deg;C</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
*Use of the&nbsp;<a href="http://tmcalculator.neb.com/#!/" target="_blank">NEB Tm&nbsp;Calculator</a>&nbsp;is highly recommended.<br /><br /><strong>General Guidelines:</strong><br /><br />1. Template:<br />Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 &micro;l reaction are as follows:
<table cellspacing="0" cellpadding="1">
<tbody>
<tr><th>DNA</th><th>AMOUNT</th></tr>
<tr>
<td>DNA Genomic</td>
<td>1 ng&ndash;1 &micro;g</td>
</tr>
<tr>
<td>Plasmid or Viral</td>
<td>1 pg&ndash;1 ng</td>
</tr>
</tbody>
</table>
<br />2. Primers:<br />Oligonucleotide primers are generally 20&ndash;40 nucleotides in length and ideally have a GC content of 40&ndash;60%. Computer programs such as&nbsp;<a class="ga-event" href="http://frodo.wi.mit.edu/primer3" target="_new" data-ga-category="External Link" data-ga-action="https://www.neb.com/protocols/2012/08/29/protocol-for-q5-high-fidelity-2x-master-mix-m0492" data-ga-label="http://frodo.wi.mit.edu/primer3">Primer3</a>&nbsp;can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 &micro;M in the reaction.<br /><br />3. Mg++&nbsp;and additives:&nbsp;<br />The&nbsp;Q5 High-Fidelity Master Mix contains 2.0&nbsp;mM Mg++&nbsp;when used at a 1X concentration. This is optimal for most PCR products generated with this master mix.&nbsp;<br /><br />4. Deoxynucleotides:<br />The final concentration of dNTPs is 200 &mu;M of each deoxynucleotide in the 1X&nbsp;Q5 High-Fidelity Master Mix.&nbsp;Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or templates.<br /><br />5. Q5&nbsp;High-Fidelity DNA Polymerase concentration:<br />The concentration of&nbsp;Q5 High-Fidelity DNA Polymerase in the&nbsp;Q5 High-Fidelity 2X Master Mix has been optimized for best results under a wide range of conditions.&nbsp;<br /><br />6. Denaturation:<br />An initial denaturation of 30 seconds at 98&deg;C is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that require it.<br /><br />During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5&ndash;10 second denaturation at 98&deg;C is recommended for most templates.<br /><br />7. Annealing:<br />Optimal annealing temperatures for&nbsp;Q5 High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. The&nbsp;<a href="https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator">NEB Tm&nbsp;Calculator</a>&nbsp;should be used to determine the annealing temperature when using this enzyme. Typically use a 10&ndash;30 second annealing step at 3&deg;C above the Tm&nbsp;of the lower Tm&nbsp;primer. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.<br /><br />For high Tm&nbsp;primer pairs, two-step cycling without a separate annealing step can be used (see note 10).&nbsp;<br /><br />8. Extension:<br />The recommended extension temperature is 72&deg;C. Extension times are generally 20&ndash;30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid,&nbsp;E. coli, etc.) or complex templates &lt; 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.<br /><br />A final extension of 2 minutes at 72&deg;C is recommended.&nbsp;<br /><br />9. Cycle number:<br />Generally, 25&ndash;35 cycles yield sufficient product. &nbsp;For genomic amplicons, 30-35 cycles are recommended.&nbsp;<br /><br />10. 2-step PCR:<br />When primers with annealing temperatures &ge;&nbsp;72&deg;C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.&nbsp;<br /><br />Amplification of long products:<br />When amplifying products &gt; 6 kb, it is often helpful to increase the extension time to 40&ndash;50 seconds/kb.&nbsp;<br /><br />11. PCR product:<br />The PCR products generated using&nbsp;Q5 High-Fidelity&nbsp;2X&nbsp;Master Mix&nbsp;have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as&nbsp;Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated.<br /><br />Addition of an untemplated -dA can be done with&nbsp;Taq&nbsp;DNA Polymerase (<a href="https://www.neb.com/products/m0267-taq-dna-polymerase-with-thermopol-buffer" target="_blank">NEB #M0267</a>&nbsp;) or Klenow exo&ndash;&nbsp;(<a href="https://www.neb.com/products/m0212-klenow-fragment-3-5-exo" target="_blank">NEB #M0212</a>&nbsp;).

[STEPS]
?. 
?. 
?. 
?.