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[DESCRIPTION]
PCR Protocol for&nbsp;Taq&nbsp;DNA Polymerase with ThermoPol&reg;&nbsp;Buffer (M0267)

[GUIDELINES]
<strong>OVERVIEW</strong><br /><br />The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification (1).&nbsp;Taq&nbsp;DNA Polymerase is an enzyme widely used in PCR (2). The following guidelines are provided to ensure successful PCR using&nbsp;NEB's&nbsp;Taq&nbsp;DNA Polymerase. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure, low template concentrations, or amplicons greater than 5 kb may require further optimization.<br /><br /><strong>PROTOCOL</strong><br /><br /><strong>Reaction setup:&nbsp;</strong><br /><br />We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95&deg;C).<table><tbody><tr><td><strong>Component</strong></td><td><strong>25 &mu;l reaction</strong></td><td><strong>50 &mu;l reaction</strong></td><td><strong>Final Concentration</strong></td></tr><tr><td>10X ThermoPol Reaction Buffer</td><td>2.5 &micro;l</td><td>5 &mu;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 &mu;l</td><td>200 &micro;M</td></tr><tr><td>10 &micro;M Forward Primer</td><td>0.5 &micro;l</td><td>1 &mu;l</td><td>0.2 &micro;M (0.05&ndash;1 &micro;M)</td></tr><tr><td>10 &micro;M Reverse Primer</td><td>0.5 &micro;l</td><td>1 &mu;l</td><td>0.2 &micro;M (0.05&ndash;1 &micro;M)</td></tr><tr><td>Template DNA</td><td>variable</td><td>variable</td><td>&lt;1,000 ng</td></tr><tr><td>Taq&nbsp;DNA Polymerase</td><td>0.125 &micro;l</td><td>0.25 &micro;l</td><td>1.25 units/50 &micro;l PCR</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.<br /><br />Transfer PCR tubes from ice to a PCR machine with&nbsp;the block preheated to 95&deg;C and begin thermocycling.&nbsp;<br /><br /><strong>Thermocycling conditions for a routine PCR:&nbsp;</strong><br /><br /><table><tbody><tr><td align='center'><strong>STEP&nbsp;</strong></td><td align='center'><strong>TEMP&nbsp;</strong></td><td align='center'><strong>TIME&nbsp;</strong></td></tr><tr><td>Initial Denaturation&nbsp;</td><td align='center'>95&deg;C&nbsp;</td><td>30 seconds&nbsp;</td></tr><tr><td>30 Cycles</td><td align='center'>95&deg;C<br />45-68&deg;C<br />68&deg;C&nbsp;</td><td>15-30 seconds<br />15-60 seconds<br />1 minute/kb&nbsp;</td></tr><tr><td>Final Extension</td><td align='center'>68&deg;C&nbsp;</td><td>5 minutes&nbsp;</td></tr><tr><td>Hold</td><td align='center'>4-10&deg;C&nbsp;</td><td>&nbsp;</td></tr></tbody></table><br /><strong>General Guidelines:&nbsp;</strong><br /><br />1. Template:&nbsp;<br /><br />Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 &mu;l reaction are as follows:<table><tbody><tr><td>DNA</td><td>Amount</td></tr><tr><td>genomic</td><td>1 ng&ndash;1 &mu;g</td></tr><tr><td>plasmid or viral</td><td>1 pg&ndash;1 ng</td></tr></tbody></table><br />2. Primers:<br /><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 <a href='http://bioinfo.ut.ee/primer3-0.4.0/primer3/' target='_blank'>Primer3</a> can be used to design or analyze primers. The final concentration of each primer in a PCR reaction may be 0.05&ndash;1 &mu;M, typically 0.1&ndash;0.5&nbsp;&mu;M.&nbsp;<br /><br />3. Mg++&nbsp;and additives:&nbsp;<br /><br />Mg++&nbsp;concentration of 1.5&ndash;2.0 mM is optimal for most PCR products generated with&nbsp;Taq&nbsp;DNA Polymerase. The final Mg++&nbsp;concentration in 1X ThermoPol Reaction Buffer is 2 mM. This supports satisfactory amplification of most amplicons. However, Mg++&nbsp;can be further optimized in 0.5 or 1.0 mM increments using MgSO4&nbsp;(<a href='https://www.neb.com/products/b1003-magnesium-sulfate-mgso4-solution'>NEB# B1003</a>).&nbsp;<br /><br />Amplification of some difficult targets, like GC-rich sequences, may be improved with additives, such as DMSO (3) or formamide (4).<br /><br />4. Deoxynucleotides:&nbsp;<br /><br />The final concentration of dNTPs is typically 200 &mu;M of each deoxynucleotide.<br /><br />5. Taq&nbsp;DNA Polymerase Concentration:&nbsp;<br /><br />We generally recommend using&nbsp;Taq&nbsp;DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 &mu;l reaction). However, the optimal concentration of&nbsp;Taq&nbsp;DNA Polymerase may range from 5&ndash;50 units/ml (0.25&ndash;2.5&nbsp;units/50 &mu;l reaction) in specialized applications.&nbsp;<br /><br />6. Denaturation:&nbsp;<br /><br />An initial denaturation of 30 seconds at 95&deg;C is sufficient for most amplicons from pure DNA templates. For difficult templates such as GC-rich sequences, a longer initial denaturation of 2&ndash;4 minutes at 95&deg;C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 5 minute denaturation at 95&deg;C is recommended.&nbsp;<br /><br />During thermocycling a 15&ndash;30 second denaturation at 95&deg;C is recommended.<br /><br />7. Annealing:&nbsp;<br /><br />The annealing step is typically 15&ndash;60 seconds. Annealing temperature is based on the Tm&nbsp;of the primer pair and is typically 45&ndash;68&deg;C. Annealing temperatures can be optimized by doing a temperature gradient PCR starting 5&deg;C below the calculated Tm.&nbsp; The NEB&nbsp;<a href='https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator' target='_blank'>Tm Calculator&nbsp;</a>is recommended for calculation of an appropriate annealing temperature.<br /><br />When primers with annealing temperatures above 65&deg;C are used, a 2-step PCR protocol is possible (see #10).<br /><br />8. Extension:&nbsp;<br /><br />The recommended extension temperature is 68&deg;C. Extension times are generally 1 minute per kb. A final extension of 5 minutes at 68&deg;C is recommended.<br /><br />9. Cycle number:&nbsp;<br /><br />Generally, 25&ndash;35 cycles yields sufficient product. Up to 45 cycles may be required to detect low-copy-number targets.&nbsp;<br /><br />10. 2-step PCR:&nbsp;<br /><br />When primers with annealing temperatures above 65&deg;C are used, a 2-step thermocycling protocol is possible.<br /><br />Thermocycling conditions for a routine 2-step PCR:<br /><table><tbody><tr><td align='center'><strong>STEP&nbsp;</strong></td><td align='center'><strong>TEMP&nbsp;</strong></td><td align='center'><strong>TIME&nbsp;</strong></td></tr><tr><td>Initial Denaturation</td><td align='center'>95&deg;C</td><td>30 seconds</td></tr><tr><td>30 Cycles</td><td align='center'>95&deg;C<br />65-68&deg;C</td><td>15-30 seconds<br />1 minute/kb</td></tr><tr><td>Final Extension</td><td align='center'>65-68&deg;C</td><td>5 minutes</td></tr><tr><td valign='top'>Hold</td><td align='center' valign='top'>4-10&deg;C</td><td valign='top'>&nbsp;</td></tr></tbody></table><br />11. PCR product:&nbsp;<br /><br />The PCR products generated using&nbsp;Taq&nbsp;DNA Polymerase contain dA overhangs at the 3&acute;&ndash;end; therefore the PCR products can be ligated to dT/dU-overhang vectors.<br /><br /><strong>References:</strong><br />1.&nbsp;&nbsp;Saiki R.K. et al. (1985).&nbsp;Science. 230, 1350-1354.<br />2.&nbsp;&nbsp;Powell, L.M. et al. (1987).&nbsp;Cell. 50, 831-840.<br />3. &nbsp;Sun, Y., Hegamyer, G. and Colburn, N. (1993).&nbsp;Biotechniques. 15, 372-374.<br />4.&nbsp; Sarkar, G., Kapelner, S. and Sommer, S.S. (1990).&nbsp;Nucleic Acids Res.. 18, 7465.

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