Preliminary

• book the Cluster Station (CS);
• perform steps 1-2 of Lin.&Hyb. protocol;

Buffers

1. prepare tubes, label and store them on ice:
   • 15ml tube:
     • #3 — linearization mix;
   • 1,7ml screw-cap tubes:
     • #5 — blocking mix
     • #7 — sequencing primer mix
     • #14 — H₂O
     • #15 — blocking buffer
     • #17 — 0,1N NaOH (from the kit)
     • #18 — TE (from the kit)
   • 50ml tubes (remain from cluster preparation step):
     • #10 — wash buffer
     • #12 — storage buffer


2. fill tubes:
   • #10 — 5ml wash buffer
   • #12 — 5ml storage buffer
   • #14 — 1,5ml H₂O


3. prepare linearization mix:
   Na Periodate + 1518µl H₂O // 2x758
   --- vortex ---
   
   in the tube #3
   

   Formamide	1,5ml
   1M TrisCl, pH8	60µl
   --- mix ---
   Na Periodate solution	1437µl // 2x718,5
   --- mix ---
   APL	2,3µl
   --- mix ---
   total	3ml



4. prepare blocking buffer (tube #15):

   10x blocking buffer	140µl
   H2O	1260µl // 2x630



5. prepare blocking mix (tube #5):

   10x blocking buffer	151µl
   H2O	1359µl // 2x679,5
   130µM ddNTP's	30µl
   --- mix ---
   TdT	20µl



6. prepare sequencing primer mix (tube #7):

   hybridization buffer	1314µl // 2x657
   sequencing primer	6,6µl

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;
• ~250ml of clean H₂O;
• washing bottle with clean H₂O;
• 100ml glass beaker for rinsing ends of tubes;
• flowcell with clusters;
• amplification manifold (wich was used for cluster amplification);
• empty flowcell;
• plastic forcepts for flowcells;
• 1,7ml tube rack;

Linearization and hybridization

1. if necessary: turn on CS, wait 30 s, turn on computer, empty the waste container;
2. if necessary: WashFullProtocolLines (~15min)
   • check, that tubes 1,3,5,7,9,10,11,12,14,15,17,18 are filled with H₂O;
   • recipe window: "WashFullProtocolLines", "Start", "OK";
3. load flowcell with clusters;
4. attach amplification manifold (wich was used for amplification);
5. pump Storage buffer to check bublles and buffer flow: [12 · 60µl/min · 200µl];
6. "User Wait" after "Amplification wash" & follow messages on the screen:
• remove H₂O from reagent positions 3,5,7,10,12,14,15,17,18, Ok
  prime air 3,5,7,10,12,14,15,17,18;
• load reagents in positions 3,5,7,10,12,14,15,17,18, Ok
  prime reagents 3,14
  20°C · 1°C/s
  [3 · 15µl/min · 300µl]
  [14 · 15µl/min · 95µl]
  
  prime reagents 12,10,5,15
  20°C · 1°C/s
  [15 · 15µl/min · 90µl]
  38°C · 1°C/s
  [5 · 15µl/min · 95µl]
  9 times: [5 · 15µl/min · 5µl]
  
  20°C · 1°C/s
  [10 · 15µl/min · 95µl]
  [12 · 15µl/min · 95µl]
• CS asks about primer hybridization (NB! it is worth to start just before priming of 1G Analyser) Ok
  prime reagents 12,10,7,18,17 20°C · 1°C/s
  [17 · 15µl/min · 95µl]
  [18 · 15µl/min · 95µl]
  [7 · 15µl/min · 95µl]
  60°C · 1°C/s
  wait 15 min
  40°C · 1°C/s
  [10 · 15µl/min · 95µl]
  20°C · 1°C/s
  [12 · 15µl/min · 95µl]
7. air gap [26 · 15µl/min · 40µl];
8. unclip "reagent delivery port" & "central clamp" of amplification manifold;
9. transfer flowcell to 1G Analyser;
10. put special empty FC and attach the amplification manifold back;


after loading 1G Analyser come back and finish the run:

11. refill #14 with H₂O;
12. clean amplification manifold with water [14 · 60µl/min · 240µl];
13. air [26 · 40µl/min · 120µl];
14. remove amplification manifold & empty FC;
15. attach washing bridge;
16. pour fresh water in water-tubes *NN*
17. for all tubes in positions X = *NN*:
    • remove tube with reagent,
    • air gap [X · 240µl/min · 10µl];
    • rinse the end of the tube using washing bottle;
    • put the tube in water-tube;
18. recipe window: "WashFullProtocolLines", "Start", "OK" (about 15min);
19. close the program; turn off computer, turn off CS