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[DESCRIPTION]
This protocols is for PCR using Q5&reg; High-Fidelity DNA Polymerase (M0491)

[GUIDELINES]
<strong>Please note that protocols with Q5&reg; Hot Start 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 />Q5 Hot Start High-Fidelity DNA Polymerase is inhibited at room temperature, allowing flexible reaction setup (RT or ice).<br /><br />All components should be mixed prior to use.&nbsp;Q5 Hot Start High-Fidelity DNA Polymerase may be diluted in 1X Q5&nbsp;Reaction Buffer just prior to use in order to reduce pipetting errors.<br /><br /><table><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>5X Q5&nbsp;<br />Reaction Buffer</td><td>5 &micro;l</td><td>10 &micro;l</td><td>1X</td></tr><tr><td>10 mM <a href='https://www.neb.com/products/n0447-deoxynucleotide-dntp-solution-mix' target='_blank'>dNTPs</a></td><td>0.5 &micro;l</td><td>1 &micro;l</td><td>200 &micro;M</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>Q5 Hot Start High-Fidelity DNA Polymerase</td><td>0.25 &micro;l</td><td>0.5 &micro;l</td><td>0.02 U/&micro;l</td></tr><tr><td>5X Q5 High GC Enhancer (optional)</td><td>(5 &micro;l)</td><td>(10 &micro;l)</td><td>(1X)</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 />Q5 Hot Start High-Fidelity DNA Polymerase does not require a separate activation step. Standard&nbsp;Q5 cycling conditions are recommended.<br /><strong><br />Thermocycling Conditions for a Routine PCR:</strong><br /><table><tbody><tr><th><strong>STEP</strong></th><th><strong>TEMP</strong></th><th><strong>TIME</strong></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><tbody><tr><th><strong>DNA</strong></th><th><strong>AMOUNT</strong></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://bioinfo.ut.ee/primer3/' target='_blank' data-ga-category='External Link' data-ga-action='https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491' data-ga-label='http://bioinfo.ut.ee/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.&nbsp;<br /><br />3. Mg++&nbsp;and additives:<br />Mg++&nbsp;concentration of 2.0 mM is optimal for most PCR products generated with Q5 High-Fidelity DNA Polymerase. When used at a final concentration of 1X, the Q5 Reaction Buffer provides the optimal Mg++concentration.&nbsp;<br /><br />Amplification of some difficult targets, like GC-rich sequences, may be improved by the addition of 1X Q5 High GC Enhancer. The Q5 High GC Enhancer is not a buffer and should not be used alone. It should be added only to reactions with the Q5 Reaction Buffer when other conditions have failed.&nbsp;<br /><br />4. Deoxynucleotides:&nbsp;<br />The final concentration of dNTPs is typically 200 &mu;M of each deoxynucleotide. Q5 Hot Start High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or templates.&nbsp;<br /><br />5. Q5 Hot Start High-Fidelity DNA Polymerase concentration:<br />We generally recommend using&nbsp;Q5 Hot Start High-Fidelity DNA Polymerase at a final concentration of 20 units/ml (1.0 unit/50 &mu;l reaction). However, the optimal concentration of&nbsp;Q5 Hot Start High-Fidelity DNA Polymerase may vary from 10&ndash;40 units/ml (0.5&ndash;2 units/50 &mu;l reaction) depending on amplicon length and difficulty. Do not exceed 2 units/50 &mu;l reaction, especially for amplicons longer than 5 kb.<br /><br />6. Buffers:<br />The 5X&nbsp;Q5 Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. For difficult amplicons, such as GC-rich templates or those with secondary structure, the addition of the&nbsp;Q5 High GC Enhancer can improve reaction performance. The 5X&nbsp;Q5 Reaction Buffer contains 2.0 mM MgCl2&nbsp;at the final (1X) concentration.<br /><br />7. Denaturation:<br />Q5&nbsp;Hot Start High-Fidelity DNA Polymerase does not require a separate activation step.<br /><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. 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 />8. Annealing:<br />Optimal annealing temperatures for&nbsp;Q5 Hot Start 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' target='_blank'>NEB Tm&nbsp;Calculator</a>&nbsp;should be used to determine the annealing temperature when using this enzyme. Typically, use a 10&ndash;30 seconds 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 11).<br /><br />9. 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.<br /><br />10. Cycle number:<br />Generally, 25&ndash;35 cycles yield sufficient product. &nbsp;For genomic amplicons, 30-35 cycles are recommended.<br /><br />11. 2-step PCR:<br />When primers with annealing temperatures &ge; 72&deg;C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.<br /><br />12. 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.<br /><br />13. PCR product:<br />The PCR products generated using&nbsp;Q5 Hot Start High-Fidelity DNA Polymerase 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 Q5&nbsp;Hot Start 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>) or Klenow exo&ndash;&nbsp;(<a href='https://www.neb.com/products/m0212-klenow-fragment-3-5-exo' target='_blank'>NEB #M0212</a>).

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