Preliminary

• check the disk space (should be ### on drive D);
• wash the GAII and the table with a wet paper towel;
• perform the GAII pre-wash ("Pre-run wash" protocol);
• prepare
  • FC after primer hybridization;
  • sequencing reagents in ice;
  • 200µl and 1ml pipettes with tips;

Loading reagents

1. load reagents in the order: 1,3,4,5,7,6;
2. perform reagent priming:
   • place the bundled tube ends in the 15ml tube;
   • open "GA2_Prime_v4" recipe ⇒ Start;
   • check the waste volume (should be ~6.4ml) and record it in the run list;

FC installation

1. remove the old FC:
   • open "Manual Control/Setup" ⇒ "Load Flowcell";
   • "Instrument" ⇒ "Unlock door";
   • pump: [to: flowcell · solution: 28 · volume: 0 · asp.rate: 250 · disp.rate: 2500] ⇒ Enter;
   • lift manifolds;
   • remove the old FC, blot oil with lens cleaning tissue, wash FC with ethanol, put back into the original tube;
   • remove the prism, put it on a piece of lens cleaning tissue;
2. clean the imaging compartment to remove dust & oil with lens cleaning tissue wetted with ethanol: Peltier element, FC table, reachable metal surfaces;
3. manifolds cleaning:
   • clean manifolds with lens cleaning tissue wetted with water;
   • blot with lens cleaning tissue;
   • clean manifolds with lens cleaning tissue wetted with ethanol (only metal parts!);
   • blot with lens cleaning tissue;
4. gently wash prism and prism metal base with a stream of ethanol & blot with lens cleaning tissue;
5. change gloves;
6. prepare prism:
   • 3x clean prism with lens cleaning tissue wetted with "clean ethanol" (from 2ml aliquotes):
       ◊  ◊  ◊  wipe of prism sides with a single motion (should be a distinct sound);
       ◊  ◊  ◊  refold lens cleaning tissue;
   • 2x clean prism with dry lens cleaning tissue:
       ◊  ◊  wipe of prism sides with a single motion;
       ◊  ◊  refold lens cleaning tissue;
   • check washing quality on reflected light, repeat cleaning if necessary;
   • install the prism in the imaging compartment;
7. clean FC:
   • blot FC using lens cleaning tissue;
   • clean both sides with lens cleaning tissue wetted in water;
   • change gloves;
   • 3x clean both sides with lens cleaning tissue wetted with "clean ethanol" (from 2ml aliquotes):
       ◊  ◊  ◊  wipe of prism sides with a single motion (should be a distinct sound);
       ◊  ◊  ◊  refold lens cleaning tissue;
   • 2x clean both sides with dry lens cleaning tissue:
       ◊  ◊  wipe of prism sides with a single motion;
       ◊  ◊  refold lens cleaning tissue;
   • check washing quality on reflected light, repeat cleaning if necessary;
8. install the FC:
   • move FC to the extreme right and forward position;
   • hold the FC & slowly lower the manifolds;
   • press manifolds downward to ensure the proper contact;
9. check reagent delivery:
   • place the bundle of the waste tubings in a 1.5ml tube;
   • pump: [to: flowcell · solution: 5 · volume: 100 · asp. rate: 250 · disp. rate: 2500] ⇒ Enter;
   • check for bubbles: if bubbles appear in the inlet manifold — reinstall the FC;
   • check the volume of the waste: should be 720-880µl;
   • repeat the reagent delivery check two more times: record delivered volumes into the run list;
10. applying oil:
    • pipette about 120µl of oil, wipe the tip with lens cleaning tissue;
    • dispense the oil slowly between the FC and prism, pressing the tip against the prism as far as possible from the edge of the FC. The oil front should move with a constant speed (if not — the FC was not installed correctly and should be reinstalled);
    • check with a folded lens cleaning tissue that the oil has reached the forward-right edge of the FC;
    • check that the top of the FC is clean and there are no bubbles between the prism and FC (if necessary, remove them by lens cleaning tissue or reinstall the prism and FC);
    • close the door;

First-base incorporation

1. put waste tubings in the large waste bottle;
2. open "GA2_FirstBase_v4GA2-PEM_2x36_PE_v4" recipe ⇒ Start;
3. answer "No" for calibration;
~20min
4. click "Cancel" to pause the protocol for the focusing (see the "Focusing");

Run

1. open "GA2-PEM_2x36_PE_v4" ⇒ Start ⇒ OKresume "GA2-PEM_2x36_PE_v4" recipe;
36 cycles run takes ~2.5days
2. after run proceed with "PE Read 2" protocol;
3. after run
   • perform the post-run wash ("Post-run wash" protocol);
   • weigh reagent bottles and record results;

Notes

• the diameter of the laser beam is about the same size as the size of the picture, so the quality of the optical surfaces should be extremely high — check using reflected light;
• contact clean FC and prism only with clean(!) gloves or with lens cleaning tissue. Do not rub surfaces, clean by blotting or one-way movements;
• fold lens cleaning tissue accurately and use new clean area for every wipe;
• channels of the FC should not be dried out: contact holes with lens cleaning tissue VERY ACCURATELY (especially if FC is oriented vertically). If some channel will dry out, put a drop of High Salt Buffer on the hole with 200µl pipet — channel will be filled by capillary force;
• leakage: GAII works under negative pressure. There should be no leakage except for one during connection/disconnection of tubing. The problem associated with leakage — if the FC or FC-manifold surface is dirty, the focal plane of the glass would be disturbed — result with uneven light, bad focusing.
• bubbles disturb the focusing. Sign of problems: bubbles in the waste tubes. To localize the problem open the instrument door, pump scanning buffer through the FC (pump: [to: flowcell · solution: 3 · volume: 100 · asp. rate: 250 · disp. rate: 2500] ⇒ Enter), and try to localize in what part of the system bubbles appear. If bubbles appear in the FC input, slightly relax manifold and accurately push the flowcell in the correct position (upper-right).