TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 1">
<div class="layoutArea">
<div class="column">
<p>The SensiFAST™ SYBR® &amp; Fluorescein Kit uses a combination of the latest advances in buffer chemistry and enhancers, together with an antibody-mediated hot-start DNA polymerase system, to ensure fast, highly-specific and ultra-sensitive real-time PCR. The kit has been validated on several Bio-Rad real-time PCR instruments.</p>
<p> </p>
<p>For ease-of-use and added convenience, SensiFAST SYBR® &amp; Fluorescein is provided as a 2x mastermix containing all the components necessary for real-time PCR, including the SYBR® Green I dye, dNTPs, stabilisers and enhancers. The kit consists of a ready-to-use premix, only primers and template need to be added.</p>
</div>
</div>
</div>

[GUIDELINES]
<div class="page" title="Page 1">
<div class="layoutArea">
<div class="column">
<p><strong>Kit components</strong></p>
<p> </p>
<div class="page" title="Page 1">
<table>
<tbody>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p><strong>Reagent</strong></p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p><strong>200 x 20μL reactions</strong></p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p><strong>500 x 20μL reactions</strong></p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p><strong>2000 x 20μL reactions</strong></p>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>SensiFASTTM SYBR® &amp; Fluorescein mix (2x)</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>2 x 1 mL</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>5 x 1 mL</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>4 x 5 mL</p>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<p> </p>
<div class="page" title="Page 1">
<div class="layoutArea">
<div class="column">
<p><strong>Instrument compatibility</strong></p>
<p> </p>
<p>SensiFAST SYBR® &amp; Fluorescein Kit has been optimized for use in SYBR® Green-based real-time PCR on the real-time PCR instruments listed in the following compatibility table, each of these instruments having the capacity to analyze the real-time PCR data with the passive reference signal either on or off. The kit is also compatible with several instruments that do not require the use of ROX, such as the Mic (BMS), Qiagen (Corbett) Rotor- Gene™ 6000, the Bio-Rad CFX96 or the Roche LightCycler® 480.</p>
<p> </p>
<div class="page" title="Page 1">
<table>
<tbody>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p><strong>Manufacturer</strong></p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p><strong>Model</strong></p>
</div>
</div>
</td>
</tr>
<tr>
<td>
<div class="layoutArea">
<div class="column">
<p>Bio-Rad</p>
</div>
</div>
</td>
<td>
<div class="layoutArea">
<div class="column">
<p>iCycler®, MyiQ™, iQ®5</p>
</div>
</div>
</td>
</tr>
</tbody>
</table>
<p> </p>
<div class="page" title="Page 1">
<div class="layoutArea">
<div class="column">
<p><strong>General considerations</strong></p>
<p> </p>
<p>To help prevent any carry-over DNA contamination we recommend that separate areas are maintained for reaction set-up, PCR amplification and any post-PCR gel analysis. It is essential that any tubes containing amplified PCR product are not opened in the PCR set-up area.</p>
<p> </p>
<div class="page" title="Page 1">
<div class="layoutArea">
<div class="column">
<p><strong>Primers:</strong> The specific amplification, yield and overall efficiency of any real-time PCR can be critically affected by the sequence and concentration of the primers, as well as by the amplicon length. We strongly recommend taking the following points into consideration when designing and running your real- time PCR:</p>
<ul>
<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>
</ul>
<div class="page" title="Page 1">
<div class="layoutArea">
<div class="column">
<ul>
<li>final primer concentration of 400n M is suitable for most SYBR® -Green based reactions, however to determine the optimal concentration we recommend titrating in the range 0.1-1 μM. The forward and reverse primers concentration should be equimolar</li>
<li> when amplifying from cDNA, use of intron spanning primers is preferable, to avoid amplification from genomic DNA</li>
</ul>
<div class="page" title="Page 1">
<div class="layoutArea">
<div class="column">
<p> </p>
<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 recommended amount of template for PCR is dependent upon the type of DNA used. The following points should be considered when using genomic DNA and cDNA templates:</p>
<div class="page" title="Page 1">
<div class="layoutArea">
<div class="column">
<ul>
<li> Genomic DNA: 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 Kit (BIO-52066) for high yield and purity from both prokaryotic and eukaryotic sources.</li>
<li> cDNA: the optimal amount of cDNA to use in a single PCR is dependent upon the copy number of the target gene. We suggest using 100 ng cDNA per reaction, however it may be necessary to vary this amount. To perform a two-step RT-PCR, we recommend using the SensiFAST cDNA Synthesis Kit (BIO-65053) for reverse transcription of the purified RNA. For high yield and purity of RNA, use the Bioline ISOLATE II RNA Mini Kit (BIO-52072).</li>
</ul>
<p> </p>
<div class="page" title="Page 1">
<div class="layoutArea">
<div class="column">
<p><strong>MgCl<sub>2</sub>:</strong> The MgCl<sub>2</sub> concentration in the 1x reaction mix is 3 mM. In the majority of real-time PCR conditions this is optimal for both the reverse transcriptase and the hot-start DNA polymerase. If necessary, we suggest titrating the MgCl<sub>2</sub> to a maximum of 5 mM.</p>
<p> </p>
<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) reaction, replacing the template with PCR grade water. When performing a two-step RT-PCR, set up a no-RT control as well as an NTC for the PCR.</p>
<p> </p>
<p><strong>Optional Fluorescein well-factor correction:</strong> SYBR® Fluorescein Kit is premixed with fluorescein, so that fluorescence emitted by fluorescein can be optionally detected on certain real-time instruments. If your real-time instrument has the capability of using fluorescein and you wish to use this option, then this option must be selected by the user in the software.</p>
<p> </p>
<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/2667/sensifast_sybr_fluorescein_kit_manual.pdf" target="_blank">http://www.bioline.com/us/downloads/dl/file/id/2667/sensifast_sybr_fluorescein_kit_manual.pdf</a></p>
<p> </p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.