Buffers

1. prepare staining solution:
   staining buffer (SB):

   1M TrisCl, pH8	500µl
   H2O	4,5ml
   L(+) Ascorb. acid Na salt	99mg
   total	5ml

   • vortex, filter through 0,2 m filter;
   • 49,5µl SB + 0,5µl SybrGreen I, mix;
   • 1ml SB + 20µl SybrGreen I (1:100), mix;
2. prepare 8-strips, label and store them on ice:
   • #S — 8 x 120µl of storage buffer,
   • #W — 8 x 240µl of H₂O,
   • #Y — 8 x 120µl of staining solution
   • #O — empty

Equipment and consumables

• powder-free gloves;
• 1ml automatic pipette with tips (for emergency cases);
• 200µl automatic pipette with tips (for emergency cases);
• lens cleaning tissue;
• washing bottle with clean H₂O;
• staining manifold;
• empty flowcell;
• Petri dish;
• Parafilm

Protocol

1. turn on CS, wait 30 s, turn on computer;
2. empty the waste container;
3. wash tubing [12 · 240µl/min · 240µl];
4. air gap [26 · 40µl/min · 120µl];
5. remove previous manifold (or washing bridge);
6. load flowcell, tube strip holder, staining manifold;
7. strip #Y(staining solution) [10 · 30µl/min · 100µl];
8. strip #0(empty tube), air gap [10 · 40µl/min · 25µl];
9. unclip "reagent delivery port" & "central clamp" of staining manifold;
10. remove flowcell, accurately clean flowcell with water and lens cleaning tissue;
11. put flowcell in Petri dish, close holes with 2 pieces of parafilm;
12. put special empty FC and attach the staining manifold back;
13. analyse clusters on fluorescent microscope;
14. depending on the plans:
    
    to store the FC:
    • unclip reagent delivery port & central clamp of staining manifold;
    • remove empty FC, put flowcell with clusters;
    • 8-strip #S(storage solution) [10 · 60µl/min · 240µl];
    • 8-strip #0(empty tube), air gap [10 · 40µl/min · 25µl];
    • unclip "reagent delivery port" & "central clamp" of staining manifold;
    • accurately clean flowcell with water and lens cleaning tissue;
    • put flowcell in 50ml tube with Storage buffer, store at 4°C;
    
    
    wash staining manifold
    
    • put special empty FC and attach back amplification manifold;
    • #W(H₂O) [10 · 60µl/min · 240µl];
    • strip #0(empty tube), air [10 · 40µl/min · 120µl];
    • remove staining manifold;
    
    
    if it is necessary to wash amplification manifold
    
    • attach used amplification manifold;
    • clean used amplification manifold with water [3 · 60µl/min · 240µl];
    • air [26 · 40µl/min · 120µl];
    • remove amplification manifold & empty FC;
    • go to "CS cleaning after amplification"
    
    
    to hybridise seq. primer:
    • #W(H₂O) [10 · 60µl/min · 240µl];
    • 8-strip #0(empty tube), air [10 · 40µl/min · 120µl];
    • remove staining manifold and empty FC;
    • put flowcell with clusters and attach used amplification manifold;
    • wash with storage buffer [12 · 40µl/min · 120µl];
    • primer hybridization (go to §3)