• Home »
  • Next generation seq.
  • Terms
  • Applications
  • Schemes »
    • Fragment libr. prep
    • Mate-Paired libr. prep
    • Clonal amplification
    • Polymerization-based sequencing
    • Ligation-based sequencing
• Illumina »
  • Library »
    • Fragment
    • Mate-Paired
    • Mate-Paired Long
    • Library amplification
  • Cluster Station »
    • Cluster generation
    • Cluster control
    • FlowCell processing
    • Multiple primer hyb.
    • DECON wash
  • 2G analyzer »
    • Focusing
    • Reagents
    • Sequencing (first read)
    • Sequencing (PE read)
  • Data analysis
  • Illumina materials
• SOLiD »
  • Library »
    • Fragment
    • Mate-Paired
    • Mate-Paired Long
    • Library amplification
  • ePCR »
    • ePCR amplification
    • Bead wash
    • Bead enrichment
    • 3'-end modification
  • Sequencing »
    • Bead deposition
    • Flowcell installation
    • QC-run
    • Sequencing run
    • Enzyme and Primer plates
  • SOLiD Kits handling
  • SOLiD materials
• General »
  • Solutions
  • Consumables
  • Kits and enzymes
  • DNA »
    • RNase treatment
    • Shearing
    • Purification
  • RNA »
    • mRNA purification on Dynabeads
    • ds cDNA synthesis
  • Oligonucleotides
  • NA quantitation
  • NA electrophoresis
  • Agarose
  • PAAG
  • Data analysis »
    • UNIX comands

Flowcell installation

Equipment and consumables Installation Focusing Notes  Comments

NB! post-PCR operation.

Equipment and consumables

• check that data collected from the previous run are satisfactory;
• empty the waste canister;
• prepare:
  • two glass beakers for washing tubing;
  • three glass beakers for O-ring wash:
    

    H2O initial water wash 70% ethanol ethanol wash H2O final water wash

• aspiration pump;
• waste bin for excess of 1x Instrument Buffer;
• 2ml tubes for control of deposition;
• screwdrivers for bolts & for deposition chamber;
• place, where to put the old assembly (storage chamber or PP-container for slides);
• plastic box with water for plastic parts of deposition chambers;
• plastic box for metal parts of deposition chambers;

Installation

1. launch ICS:
   • remove previous run: "clear";
   • if necessary, unlock doors;
   • click on reagent chiller button to select "Cooling" (bottom middle);
2. create a new run (see "QC-run" or "35-bp run" or PE-run");
3. prepare instrument FlowCell:
   • insert paper towels under the FC chambers;
   • in ICS: Clear Flowcell & Yes;
   • open FlowCell chamber;
   • remove old slide assembly & put it in a appropriate place (work fast to prevent drying of slides);
   • clean O-rings:
       •   remove O-ring;
       •   THOROUGHLY check integrity of O-ring;
       •   wash O-ring sequentially by water, 70% ethanol & water again;
   • clean FlowCell chamber:
       •   remove excess of the liquid from FlowCell chamber (aspiration & paper towel);
       •   clean FlowCell chamber with water and 70% ethanol;
       •   carefully remove all liquid;
   • put few drops of 1x Instrument buffer on FlowCell chamber & install O-ring (DO NOT TWIST O-ring!);
   • check evenness of O-ring by finger, re-install it if necessary;
4. open a new slide:
   • remove adhesive paper from holes;
   • add 1x Instrument Buffer to the reservoir of deposition chamber;
   • with 1ml pipettor aspirate SLOWLY the Deposition Buffer from the well & put it into prepared 2ml tube (later control the absence of beads on magnetic stand);
   • diagonally loosen screws;
   • open assembly;
   • put additional 1x Instrument Buffer on the top of the slide;
5. install slide:
   • click "Load Flowcell" button (DO NOT click "OK");
   • find orientation of the slide carrier;
   • QUICKLY pour off the excess of the 1x Instrument Buffer & install slide carrier (work fast to prevent drying of slides);
   • gradually tighten two bolts;
   • check, that liquid does not move when tightening;
   • aspirate the excess of liquid from the FlowCell holder;
   • wipe first with wet and then with dry paper towel the glass surface;
   • rotate FlowCell up & lock;
   • click OK in the ICS dialog box;
   • wait, until FolwCell is loaded & open FlowCell down;
   • check, that there are not to much bubbles (otherwise repeat "Load Flowcell" command);
   • aspirate excess of the liquid from FlowCell block, liquid trap & clean it with 70% ethanol;
6. clean glass slide:
   • 2x one-way wiping with lens-cleaning tissue slightly moistened with water;
   • 3x one-way wiping with lens-cleaning tissue slightly moistened with ethanol;
   • rotate FlowCell up & lock;
   • repeat "Load Flowcell" command and check for leaking;





Focusing

7. find the old "average distance" in the file C:/Documents and Settings/All Users/Application Data/Applied Biosystems/discovery/imager_local (link on the desktop);
   
   old focus: ____________    ____________    ____________    ____________
8. lock doors of the instrument;
9. menu: Window > Imaging System;
10. select the following parameters (repeat the already selected):
    • proper flowcell number in "Show FlowCell";
    • "Gain": 1;
    • WL (white light) in "Filter status" box;
    • "2" in "Nosepiece Status";
    • "2" in "Prism Status";
11. menu: View > Stage template & select proper *.STG file:
    • 1 well: mask_1_spot
    • 4 wells: mask_4_spot
    • 8 wells: mask_8_spot
12. move green-box cursor into upper-left corner;
13. in "Focus Status" go to "old focus" value;
14. click "AutoExposure";
15. manually find a focus position (with 500 accuracy for EXTREME values):
    • acquire;
    • check image & correct focus distance;
determine the focus positions in corners and in centre of the FlowCell(s) 1 2 3 4 5 6 :

NB! reinstall the glass if the "MAX-MIN" range exceeds 5000
Z=(MIN+MAX)/2=________________


     _________________               _________________

                         _________________

     _________________               _________________




Z=(MIN+MAX)/2=________________


     _________________               _________________

                         _________________

     _________________               _________________




Z=(MIN+MAX)/2=________________


     _________________               _________________

                         _________________

     _________________               _________________




Z=(MIN+MAX)/2=________________


     _________________               _________________

                         _________________

     _________________               _________________




Z=(MIN+MAX)/2=________________


     _________________               _________________

                         _________________

     _________________               _________________




Z=(MIN+MAX)/2=________________


     _________________               _________________

                         _________________

     _________________               _________________




16. calculate the (MIN+MAX)/2 value;
17. menu: Options > Focusing:
    • switch off "Use default focusing range";
    • insert: Z Min = Z-5000; Z Max = Z+5000;
    • "Save to Flowcell" and approve the FlowCell number;
18. repeat §§3-11 for the flowcell 2;
19. check new settings:
    • close all windows;
    • open ICS again;
    • check that new settings are in the imager_local file;
    • menu: Window > Imaging System;

Notes

• never scrape surfaces with paper towels. To remove liquid: press paper towel, wait few seconds and remove;