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[DESCRIPTION]
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<p>The SensiFAST™ HRM Kit has been developed for fast, highly reproducible High Resolution Melt (HRM) analysis and has been validated on commonly used real-time instruments. A combination of the latest advances in buffer chemistry and enhancers, together with an antibody-mediated hot-start DNA polymerase system, ensures that the SensiFAST HRM Kit delivers fast, highly-specific and ultra-sensitive HRM analysis.</p>
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<p>For ease-of-use and added convenience, SensiFAST HRM is provided as a 2x mastermix containing all the components necessary for real-time PCR, including the EvaGreen® dye, dNTPs, stabilisers and enhancers. As a ready-to-use premix, only primers and template need to be added.</p>
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[GUIDELINES]
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<p><strong>Kit components</strong></p>
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<p><strong>Reagent</strong></p>
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<p><strong>200 x 20 μL reactions</strong></p>
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<p><strong>500 x 20 μL reactions</strong></p>
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<p><strong>2000 x 20 μL reactions</strong></p>
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<p>SensiFAST HRM mix (2x)</p>
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<p>2 x 1 mL</p>
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<p>5 x 1 mL</p>
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<p>20 x 1 mL</p>
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<p><strong>General considerations</strong></p>
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<p>To help prevent any carry-over DNA contamination, we recommend that separate areas be maintained for PCR set-up, PCR amplification and any post-PCR gel analysis. It is essential that any amplified PCR product should not be opened in the PCR set-up area.</p>
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<p><strong>Primers:</strong> The sequence and concentration of the primers, as well as amplicon length, can be critical for specific amplification, yield and overall efficiency of any real-time PCR. We strongly recommend taking the following points into consideration when designing and running your real-time PCR:</p>
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<li>use primer-design software, such as Primer3 (<a href="http:// frodo.wi.mit.edu/primer3/" target="_blank">http:// frodo.wi.mit.edu/primer3/</a>) or visual OMP™ (<a href="http:// dnasoftware.com/" target="_blank">http:// dnasoftware.com/</a>). Primers should have a melting temperature (Tm) of approximately 60°C.</li>
<li>optimal amplicon length should be 80-200 bp, and should not exceed 400 bp</li>
<li>final primer concentration of 400 nM is suitable for most reactions, however to determine the optimal concentration we recommend titrating in the range 0.1-1 μM</li>
<li>use an equimolar primer concentration</li>
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<p><strong>Template:</strong> it is important that the DNA template is suitable for use in PCR in terms of purity and concentration. In addition, the template needs to be devoid of any contaminating PCR inhibitors (e.g. EDTA). The following should be considered when using genomic DNA templates:</p>
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<li><strong>Genomic DNA</strong>: use up to 1 μg of complex (e.g. eukaryotic) genomic DNA in a single PCR. We recommend using the Bioline ISOLATE II Genomic DNA Mini Kit (BIO-52066) for high yield and purity from both prokaryotic and eukaryotic sources</li>
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<p><strong>MgCl<sub>2</sub>:</strong> The MgCl<sub>2</sub> concentration in the 1x reaction mix is 3 mM, which is optimal for SensiFAST HRM in the majority of real-time PCR conditions. If necessary, we suggest titrating MgCl<sub>2 </sub>to a maximum of 5 mM.</p>
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<p><strong>PCR controls:</strong> It is important to detect the presence of contaminating DNA that may affect the reliability of the data. Always include a no template control (NTC), replacing the template with PCR-grade water.</p>
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<p><strong>Troubleshooting Guide</strong></p>
<p>See the Bioline full documentation for detailed troubleshooting instructions.</p>
<p><a href="http://www.bioline.com/us/downloads/dl/file/id/2705/sensifast_hrm_kit_manual.pdf" target="_blank">http://www.bioline.com/us/downloads/dl/file/id/2705/sensifast_hrm_kit_manual.pdf</a></p>
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