Priming lines (20 µl per one step)

• 1-4, 9-18: 17 cycles
• 5-8: 13 cycles

Work area

• clean work:
  • powder-free gloves;
  • lens cleaning tissue;
  • 1L bottle with mQ water;
  • washing bottle with mQ water;
  • 100ml glass beaker for rinsing tubes;
• boxes:
  • for storage of tube caps;
  • for Washing bridge storage;
• instruments:
  • plastic tweezers;
  • empty flowcell for washing of used manifolds;
  • 1ml and 200µl tips;
  • tubes: 50ml, 15ml, 2ml; 8-strips;
  • tube holders: 50ml, 2ml;
• cluster staining:
  • staining manifold;
  • Petri dishes for stained FCs;
  • pieces of parafilm for stained FCs;
• writing:
  • marker;
  • pen;

Flowcell (FC)

• contact only with clean(!) gloves or with lens cleaning tissue;
• channels should not be dried out: contact WET holes with lens cleaning tissue VERY ACCURATELY!!! Especially if FC is oriented vertically;
• leakage: Both CS and 1GA work under the negative pressure. So, there should be no leakage except for one during connection/disconnection of tubing. Real problems: blockage of one of the channels or bubbles in the line.

Manifolds

• tubes ~2.85µl/cm
  • amplification & hybridization manifolds: 2x[13cm · 37µl];
  • washing bridge [30cm · 86µl]
• flowrate restrictions:
  • washing bridge: < 240µl/min;
  • flowcell: < 60µl/min;
  • to avoid small droplets in tubing: < 30µl/min

Washing of manifolds

1. if necessary, remove the FlowCell:
   • air gap

     manifold program amplification 26 · 30µl/min · 40µl hybridization 26 · 30µl/min · 25µl washing bridge 26 · 30µl/min · 120µl

   • unclip "reagent delivery port" & "central clamp";
   • raise up manifold;
   • remove FlowCell;
2. insert an empty FlowCell and attach the manifold;
3. wash with water:
   • amplification manifold: [x · 60µl/min · 200µl], x is a position of H₂O tube;
   • hybridisation/staining manifold: [26 · 60µl/min · 200µl] from a 8-strip with 240µl H₂O per tube;
4. air gap:
   • amplification manifold: [26 · 40µl/min · 120µl];
   • hybridisation/staining manifold: [26 · 40µl/min · 120µl] from empty 8-strip;
5. unclip the manifold;
6. clean the rubber parts with (i) wet and (ii) dry lens cleaning tissue;
7. put manifold into container;
8. write date on the container;

Unloading of manifolds

1. air gap:

   manifold program amplification 26 · 30µl/min · 40µl hybridization 26 · 30µl/min · 25µl washing bridge 26 · 30µl/min · 120µl

2. unclip from "left" to "right":
   • reagent delivery port,
   • central clamp,
   • waste port;
3. remove FlowCell if necessary;
4. wipe the ports and the flowcell stage with water and dry with lens cleaning tissue.

Maintenance

• every 2 weeks: DECON wash;
• after each usage: wash (i) used tubing and (ii) manifolds with H₂O, (iii) empty the waste bottle;
• before each usage:
  • clean flowcell area with H₂O and lens cleaning tissues;
  • if CS was previously used by:
    • our group: change water in water storage tubes (WST);
    • another group: replace WSTs with tubing washing and air priming;
      
    • another group (DECON wash):
           50ml & 15ml tubes: shake the old WSTs gently to wash the cap; change water in WSTs and shake again; remove drops of water from the caps by 1ml pipette; replace WSTs with tubing washing and air priming;
           2ml tubes: wash caps twice with water; replace WSTs with tubing washing and air priming;