Preliminary

• book the Cluster Station (CS);
• prepare Cluster Generation Kit Box 2 (NT);
• thaw sequencing primers;

Prepare reagents

NB! store all components on ice

1. label strips and store them on ice:

   #F	NaOH, 0.1N
   #G	TE buffer
   #H	primer mixture
   #I	wash buffer
   #J1	storage buffer
   #J2	storage buffer
   #O	empty
   #W	H2O for washing manifold



2. prepare primer mix in hybridization buffer (recommended dilution at least 1:200):
   
   number of channels:

   conc. stock primer 0.6µM hybridization buffer



3. fill strips:

   #F	100µl of NaOH, 0.1N
   #G	150µl of TE buffer
   #H	100µl of primer mixture
   #I	100µl of wash buffer
   #J1 & #J2	100µl of storage buffer
   #W	240µl of H2O



4. take following things to CS:
   • flowcell with clusters;
   • amplification manifold (from the cluster generation step);
   • storage buffer (for the case of problems with manifold);
   • 1ml and 200µl pipettes;

Primer hybridization

1. if necessary: turn on CS, wait 30 s, turn on computer, empty the waste container;
2. wipe the table and the CS with a wet paper towel;
3. open "MultiplePrimerhyb_only_v3.0" recipe;
4. install flowcell:
   • remove liquid from the washing bridge [26 · 40µl/min · 120µl];
   • remove washing bridge;
   • install flowcell and hybridization manifold;
5. put the #J1 strip with storage buffer in the strip holder;
6. pump storage buffer and check (IMPORTANT!) the liquid flow through the all channels [26 · 60µl/min · 100µl];
7. remove the #J1 strip and pump a small air gap [26 · 40µl/min · 10µl];
"User Wait" after the washing step


8. follow messages on the screen:
   • install the FC and the hybr.manifold ⇒ OK;
   • strip #F ⇒ OK
     [26 · 15µl/min · 75µl]
     
   • strip #G ⇒ OK
     [26 · 15µl/min · 75µl]
     
   • strip #H ⇒ OK
     [26 · 15µl/min · 75µl]
        60°C · 1°C/s
        wait 15 min
        40°C · 1°C/s
     
   • strip #I ⇒ OK
     [26 · 15µl/min · 75µl]
        20°C · 1°C/s
     
   • strip #J2 ⇒ OK
     [26 · 15µl/min · 75µl]
     


9. remove FC:
   • strip #O;
   • air gap [26 · 15µl/min · 40µl];
   • unclip "reagent delivery port" & "central clamp" of hybridization manifold;
   • remove FC and accurately clean it with water and lens cleaning tissue;
   • transfer flowcell to 1G Analyser;
   • install empty FC and attach the hybridization manifold back;


10. wash hybridization manifold (see "Washing of manifolds");
11. close the program; turn off computer, turn off CS.

Notes

• Volumes of reagents after reaction (per tube):

  position reagent initial volume should remain #F NaOH, 0.1N 100µl 25µl #G TE buffer 150µl 75µl #H seq. primer mix 100µl 25µl #I wash buffer 100µl 25µl #J1 & #J2 storage buffer 100µl 25µl