TITLE: No Title
AUTHORS: 

[DESCRIPTION]
OneTaq&nbsp;DNA Polymerase allows for greater amplification sensitivity across a wide variety of amplicons regardless of GC content.&nbsp;

[GUIDELINES]
<strong>OVERVIEW<br /><br />PCR&nbsp;</strong><br /><br />The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification.&nbsp;Taq&nbsp;DNA Polymerase is an enzyme widely used in PCR. OneTaq&nbsp;DNA Polymerase allows for greater amplification sensitivity across a wide variety of amplicons regardless of GC content. The following guidelines are provided to ensure successful PCR using New England Biolabs&rsquo; OneTaq&nbsp;DNA Polymerase. These guidelines cover most routine PCR. Specialized applications 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 (94&deg;C).<br /><br />Add to a sterile thin-walled PCR tube:<br /><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>5X OneTaq&nbsp;Standard&nbsp;<br />Reaction Buffer*</td><td>5 &micro;l</td><td>10 &mu;l</td><td>1X</td></tr><tr><td>10 mM dNTPs (<a href='https://www.neb.com/products/n0447-deoxynucleotide-dntp-solution-mix' target='_blank'>#N0447</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&nbsp;&micro;l</td><td>1&nbsp;&mu;l</td><td>0.2 &micro;M</td></tr><tr><td>10 &micro;M Reverse Primer</td><td>0.5&nbsp;&micro;l</td><td>1&nbsp;&mu;l</td><td>0.2 &micro;M</td></tr><tr><td>OneTaq&nbsp; DNA Polymerase</td><td>0.125 &micro;l</td><td>0.25 &micro;l</td><td>1.25 units/50 &micro;l&nbsp;<br />PCR**</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>*OneTaq&nbsp;GC Reaction Buffer and High GC Enhancer can be used for difficult amplicons<br /><br />**For amplicons between 3&ndash;6 kb, use 2.5&ndash;5 units/50 &micro;l rxn<br /><br />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 to a PCR machine and begin thermocycling:<br /><br />Thermocycling conditions for a routine PCR:&nbsp;<br /><br /><table><tbody><tr><td align='center'>STEP&nbsp;</td><td align='center'>TEMP&nbsp;</td><td align='center'>TIME&nbsp;</td></tr><tr><td>Initial Denaturation</td><td align='center'>94&deg;C</td><td>30 seconds</td></tr><tr><td>30 Cycles</td><td align='center'>94&deg;C<br />45-68&deg;C<br />68&deg;C</td><td>15-30 seconds<br />15-60 seconds<br />1 minute per kb</td></tr><tr><td>Final Extension</td><td align='center'>68&deg;C</td><td>5 minutes</td></tr><tr><td>Hold</td><td align='center'>4-10&deg;C</td><td>&nbsp;</td></tr></tbody></table><br />GENERAL GUIDELINES<br /><br /><strong>Template:&nbsp;</strong><br /><br />1. 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:<br /><table><tbody><tr><td><strong>DNA</strong></td><td><strong>Amount</strong></td></tr><tr><td>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 /><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 PrimerSelect&trade; (DNAStar Inc., Madison, WI) and&nbsp;<a class='ga-event' href='http://frodo.wi.mit.edu/primer3' target='_blank' data-ga-category='External Link' data-ga-action='https://www.neb.com/protocols/2012/10/11/onetaqdnapolymerasem0480' data-ga-label='http://frodo.wi.mit.edu/primer3'>Primer3&nbsp;</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 &micro;M, typically 0.2 &micro;M.<br /><br />3. Mg++&nbsp;and Additives:<br /><br />Mg++&nbsp;concentration of 1.5&ndash;2.0 mM is optimal for most PCR products generated with OneTaq&nbsp;DNA Polymerase. The final Mg++&nbsp;concentration in 1X OneTaq&nbsp;Standard Reaction Buffer is 1.8&nbsp;mM. This supports satisfactory amplification of most amplicons. However, Mg++&nbsp;can be further optimized in 0.2 mM increments using MgCl2(<a href='https://www.neb.com/products/b9021-magnesium-chloride-mgcl2-solution'>NEB #B9021</a>). OneTaq&nbsp;(Mg-Free) Standard Reaction Buffer (<a href='https://www.neb.com/products/b9024-onetaq-mg-free-standard-reaction-buffer-pack'>NEB #B9024</a>) and supplemental MgCl2&nbsp;are also available separately for complete control of Mg++&nbsp;concentration in the reaction.<br /><br />Amplification of some difficult targets, like GC-rich sequences, may be improved by the use of OneTaq&nbsp;GC Reaction Buffer. The final Mg++&nbsp;concentration in 1X OneTaq&nbsp;GC Reaction Buffer is 2.0 mM. To optimize the Mg++&nbsp;concentration of the OneTaq&nbsp;GC Reaction Buffer, MgSO4&nbsp;should be used (<a href='https://www.neb.com/products/b1003-magnesium-sulfate-mgso4-solution'>NEB #B1003</a>). OneTaq&nbsp;(Mg-Free) GC Reaction Buffer (<a href='https://www.neb.com/products/b9025-onetaq-mg-free-gc-reaction-buffer-pack'>NEB #B9025</a>) and supplemental MgSO4&nbsp;are also available separately for complete control of Mg++&nbsp;concentration in the reaction.<br /><br />For extremely difficult amplicons, 10&ndash;20% OneTaq&nbsp;High GC Enhancer can be added to reactions with OneTaqGC Reaction Buffer. The enhancer should not be used alone and typically increases yields when other conditions have failed.<br /><br />4. Deoxynucleotides:<br /><br />The final concentration of dNTPs is typically 200 &micro;M of each deoxynucleotide.<br /><br />5. OneTaq&nbsp;DNA Polymerase Concentration:<br /><br />We generally recommend using OneTaq&nbsp;DNA Polymerase at a concentration of 25 units/ml (1.25 units/50 &micro;l reaction) for amplicons up to 3 kb. The optimal concentration of OneTaq&nbsp;DNA Polymerase may range from 5&ndash;100 units/ml (0.25&ndash;5 units/50 &micro;l reaction). For specialized applications, including 3&ndash;6 kb amplicons, 2.5&ndash;5&nbsp;units/50 &micro;l reaction is recommended. Note that in some cases increasing the amount of enzyme in the reaction can be inhibitory.<br /><br />6. Denaturation:<br /><br />An initial denaturation of 30 seconds at 94&deg;C is sufficient to amplify most targets from pure DNA templates. For difficult templates such as GC-rich sequences, a longer denaturation of 2&ndash;4 minutes at 94&deg;C is recommended prior to PCR cycling to fully denature the template. With colony PCR, an initial 2&ndash;5 minute incubation at 94&deg;C is recommended to lyse cells.<br /><br />During thermocycling a 15&ndash;30 second denaturation at 94&deg;C is recommended.<br /><br />7. Annealing:<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. The NEB&nbsp;<a href='https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator'>Tm&nbsp;Calculator</a>&nbsp;is recommended for calculation of an appropriate annealing temperature.<br /><br />8. Extension:<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:<br /><br />Generally, 25&ndash;35 cycles yield sufficient product. Up to 45 cycles may be required to detect low copy number targets.<br /><br />10. 2-step PCR:<br /><br />When primers with annealing temperatures of 68&deg;C or above are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.<br /><br />11. PCR Product:<br /><br />A significant portion of the PCR products generated using OneTaq&nbsp;DNA Polymerase contain dA overhangs at the 3&acute;end; therefore the PCR products can be ligated to dT/dU-overhang vectors.

[STEPS]
?. 
?. 
?. 
?.