http://bmcwiki.mit.edu/index.php?action=history&feed=atom&title=BioMicroCenter%3ACluster_Visualization_Protocol_for_Flowcell_QC 2026-04-29T03:17:22Z Revision history for this page on the wiki MediaWiki 1.43.1 http://bmcwiki.mit.edu/index.php?title=BioMicroCenter:Cluster_Visualization_Protocol_for_Flowcell_QC&diff=35&oldid=prev 2009-04-10T21:10:04Z

<p><span class="autocomment">Cluster Visualization for Flowcell QC</span></p> <p><b>New page</b></p><div>{{BioMicroCenter}}<br /> <br /> == Cluster Visualization for Flowcell QC ==<br /> <br /> This protocol is a slightly modified version of the protocol supplied by Illumina, and allows for the visualization of clusters on a flowcell before it is put on the Genome Analyzer. It is especially helpful to ensure proper amplification has occurred if there has been a clog or an error on the cluster station or if an older Cluster Generation kit that may be expired is being used. <br /> <br /> This protocol is to be carried out on the Cluster Station after amplification.<br /> <br /> == Materials ==<br /> <br /> * SYBR Green mix (We use Roche LightCycler 480 SYBR Master Mix, Cat. No. 04707516001)<br /> <br /> * Illumina Wash Buffer<br /> <br /> * Illumina Storage Buffer <br /> <br /> * Fluorescent microscope equipped with a GFP filter set<br /> <br /> == Protocol ==<br /> <br /> * Dilute 500uL of SYBR Green in 1500uL Wash Buffer in a 2mL microcentrifiuge tube labeled as #8 and load onto the Cluster Station<br /> <br /> * Manually prime solution 8x10 and then manually pump the dilute SYBR using the following settings:<br /> <br /> #Reagent: 8<br /> #Flowrate: 30<br /> #Volume 150<br /> <br /> * Remove the Flowcell from the cluster station for observation on the Fluorescent microscope. Use the GFP filter set, blue light. Neutral Denstity filters can be used to help prevent photobleaching<br /> <br /> &#039;&#039;&#039;If amplification was successful and clusters were observed continue with protocol. If clusters were not visualized re-cluster samples on a new Flowcell&#039;&#039;&#039;<br /> <br /> * Place Flowcell back onto Cluster Station<br /> <br /> * Label a 50mL tube as #10 and fill with about 5mL Wash Buffer. Load #10 onto the Cluster station.<br /> <br /> * Manually pump #10 using the following settings:<br /> <br /> #Reagent: 10<br /> #Flowrate: 30<br /> #Volume: 200<br /> <br /> * Label a 50mL tube as #12 and fill with about 5mL Storage Buffer. Load #12 onto the Cluster Station.<br /> <br /> * Manually pump #12 using the following settings:<br /> <br /> #Reagent: 10<br /> #Flowrate: 30<br /> #Volume: 200<br /> <br /> * Remove Flowcell and store at 4C until ready for use<br /> <br /> * Replace #8 with 1.5mL screw cap tube filled with water<br /> <br /> * Remove amplification manifold and replace with wash bridge<br /> <br /> * Was line 8 by manually pumping water using the following settings:<br /> <br /> #Reagent: 8<br /> #Flowrate: 30<br /> #Volume: 150</div>

imported>Allison Perrotta