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[DESCRIPTION]
This is the protocol for the Q5&reg; Site-Directed Mutagenesis Kit (E0554)

[GUIDELINES]
<strong>DESCRIPTION</strong><br /><br />The Q5&reg; Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours (Figure 1). The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions in a wide variety of plasmids. After PCR, the amplified material is added directly to a unique Kinase-Ligase-DpnI (KLD) enzyme mix for rapid (5 minutes), room temperature circularization and template removal (Figure 2). Transformation into high-efficiency NEB 5-alpha Competent&nbsp;E. coli, provided with the kit, ensures robust results with plasmids up to at least 20 kb in length.<br /><br /><img src="http://i.imgur.com/XO5jxp3.jpg" alt="" width="558" height="184" /><br /><strong><em>Figure 1: Site-specific mutagenesis proceeds in less than 2 hours.</em></strong><br /><em>The use of a master mix, a unique multi-enzyme KLD enzyme mix, and a fast polymerase ensures that, for most&nbsp;plasmids, the mutagenesis reaction is complete in less than two hours.<br /><br /><img src="http://i.imgur.com/hHFLxl6.jpg" alt="" width="559" height="317" /><br /><br /><strong>Figure 2: Q5 Site-Directed Mutagenesis Kit Overview.</strong><br />This kit is designed for rapid and efficient incorporation of insertions, deletions and substitutions into doublestranded&nbsp;plasmid DNA. The first step is an exponential amplification using standard primers and a master mix fomulation of Q5 Hot Start High-Fidelity DNA Polymerase. The second step involves incubation with a unique&nbsp;enzyme mix containing a kinase, a ligase and DpnI. Together, these enzymes allow for rapid circularization of the&nbsp;PCR product and removal of the template DNA. The last step is a high-efficiency transformation into chemicallycompetent&nbsp;cells (provided).<br /><br /><img src="http://i.imgur.com/wwnS5D0.jpg" alt="" width="530" height="347" /><br /><strong>Figure 3: Primer Design for the Q5 Site-Directed Mutagenesis Kit</strong><br />Substitutions, deletions and insertions are incorporated into plasmid DNA through the use of specifically designed&nbsp;forward (black) and reverse (red) primers.&nbsp;<strong>Unlike kits that rely on linear amplification, primers designed&nbsp;for the Q5 Site-Directed Mutagenesis Kit should not overlap to ensure that the benefits of&nbsp;</strong><strong>exponential amplification are realized.&nbsp;</strong>A) Substitutions are created by incorporating the desired nucleotide&nbsp;change(s) (denoted by *) in the center of the forward primer, including at least 10 complementary nucleotides on&nbsp;the 3&acute;side of the mutation(s). The reverse primer is designed so that the 5&acute; ends of the two primers anneal back-to-&nbsp;back. B) Deletions are engineered by designing standard, non-mutagenic forward and reverse primers that flank&nbsp;the region to be deleted. C) Insertions less than or equal to 6 nucleotides are incorporated into the 5&acute; end of the&nbsp;forward primer while the reverse primer anneals back-to-back with the 5&acute; end of the complementary region of the&nbsp;forward primer. D) Larger insertions can be created by incorporating half of the desired insertion into the 5&acute; ends&nbsp;of both primers. The maximum size of the insertion is largely dictated by oligonucleotide synthesis limitations.<br /></em><br /><strong>TROUBLESHOOTING<br /><br />No/Low Colonies</strong><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Ensure that your primers are designed properly. To take advantage of the exponential nature of the amplification reaction, the 5&acute; ends of the two primers should align back-to-back unless deletions are being made (see Figure 3). For best results, primers should be designed and annealing temperatures calculated using&nbsp;</span><a style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;" href="http://nebasechanger.neb.com/" target="_blank">NEBaseChanger&trade;</a><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">, the NEB online primer design software.</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Ensure there is a clean PCR product by visualizing 2&ndash;5 &mu;l of the reaction on an agarose gel. Follow the suggestions below for low or impure PCR products.</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Only use 1 &mu;l of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. If the PCR yield is low, more product can be added to the KLD reaction, however a buffer exchange step, such as PCR purification, must be included prior to transformation.</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Only use 5 &mu;l of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Ensure that the selectable marker in the plasmid matches the selection agent used in the plates</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Ensure the NEB 5-alpha Competent&nbsp;E. coli&nbsp;cells have been stored at -80&deg; C.</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Check that the transformation efficiency of the competent cells is ~1 x 109 colony forming units (cfu) per &mu;g. To calculate transformation efficiency, transform 2 &mu;l of the provided control pUC19 DNA (100 pg) into 50 &mu;l of cells. Follow the transformation protocol on page 8. Prior to plating, dilute 10 &mu;l of cells up to 1 ml in SOC. Plate 100 &mu;l of this dilution. In this case, 150 colonies will yield a transformation efficiency of 1.5 x 109&nbsp;cfu/&mu;g&nbsp;</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">(&mu;g DNA=0.0001, dilution=10/1000 x 100/1000).</span></li>
</ul>
<br /><strong>No/Low PCR Product</strong><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Ensure that the optimal annealing temperature (Ta) is used.&nbsp;</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">High-Fidelity polymerases benefit from a Tm+3 annealing temp. Use&nbsp;</span><a style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;" href="http://nebasechanger.neb.com/" target="_blank">NEBaseChanger&trade;</a><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">, the NEB online primer design software, to calculate Ta. Alternatively, the optimal annealing temperature could be determined using a gradient PCR followed by agarose gel analysis.</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Ensure that the elongation time is adequate for the plasmid length. We recommend 10&ndash;20 seconds per kb of plasmid.</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Ensure that the final concentration of each primer is 0.5 &mu;m.&nbsp;</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Purify the primers with polyacrylamide gel electrophoresis (PAGE).</span></li>
</ul>
<br /><strong>Resulting Plasmids Do Not Contain the Desired Mutation</strong><br />
<ul style="list-style-type: circle;">
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Ensure proper design of the mutagenic primers.</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Optimize the PCR conditions (see above).</span></li>
<li><span style="font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 13px; line-height: 20px;">Use 1&ndash;25 ng of template in the PCR step. A small increase in the number of clones with no/incorrect mutation incorporated can occur if less than 1 ng or more than 25 ng of template is used.</span></li>
</ul>
<br /><strong>NOTES<br />Storage Note:<br />The Q5 Site-Directed Mutagenesis Kit is stable at &ndash;80&deg;C for one year. For convenience, the Q5 Hot Start High-Fidelity 2X Master Mix, KLD Enzyme Mix, KLD Reaction Buffer, Control Primers and Template DNA are packaged together in a separate box that can be removed and stored at &ndash;20&deg;C for two years with no loss of activity. The SOC can be removed and stored at room temperature. It is important to store the NEB 5-alpha Competent&nbsp;E. coli&nbsp;at &ndash;80&deg;C, and avoid repeated freeze-thaw cycles.<br /><br /></strong>REFERENCES<br />1. Kalnins et al., (1983). The EMBO Journal. 2, 593-597.<br />2. Dickinson DJ, Ward JD, Reiner DJ, Goldstein B. (2013). Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination.. Nat Methods. Sep 1, PubMedID: 23995389

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