BioMicroCenter:Cluster Visualization Protocol for Flowcell QC

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Cluster Visualization for Flowcell QC

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.

This protocol is to be carried out on the Cluster Station after amplification.

Materials

  • SYBR Green mix (We use Roche LightCycler 480 SYBR Master Mix, Cat. No. 04707516001)
  • Illumina Wash Buffer
  • Illumina Storage Buffer
  • Fluorescent microscope equipped with a GFP filter set

Protocol

  • Dilute 500uL of SYBR Green in 1500uL Wash Buffer in a 2mL microcentrifiuge tube labeled as #8 and load onto the Cluster Station
  • Manually prime solution 8x10 and then manually pump the dilute SYBR using the following settings:
  1. Reagent: 8
  2. Flowrate: 30
  3. Volume 150
  • 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

If amplification was successful and clusters were observed continue with protocol. If clusters were not visualized re-cluster samples on a new Flowcell

  • Place Flowcell back onto Cluster Station
  • Label a 50mL tube as #10 and fill with about 5mL Wash Buffer. Load #10 onto the Cluster station.
  • Manually pump #10 using the following settings:
  1. Reagent: 10
  2. Flowrate: 30
  3. Volume: 200
  • Label a 50mL tube as #12 and fill with about 5mL Storage Buffer. Load #12 onto the Cluster Station.
  • Manually pump #12 using the following settings:
  1. Reagent: 10
  2. Flowrate: 30
  3. Volume: 200
  • Remove Flowcell and store at 4C until ready for use
  • Replace #8 with 1.5mL screw cap tube filled with water
  • Remove amplification manifold and replace with wash bridge
  • Was line 8 by manually pumping water using the following settings:
  1. Reagent: 8
  2. Flowrate: 30
  3. Volume: 150
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