Preliminary

Prepare reagents

1. [  ]  label:
   • 8-strips:

     #A	hybridization buffer
     #B	template (write the number of library on each tube)
     #C	wash buffer
     #D	amplif. premix
     #E	extension mix
     #0	empty

   
   
   • 1.7ml tube:

     #IEM	initial extension mix

   
   
   • 50ml tubes:

     #1	Bst mix
     #9	formamide
     #10	wash buffer
     #11	ampl. premix
     #12	storage buffer



2. [  ]  fill the tubes:

   #9	15ml formamide
   #10	10ml wash buffer
   #12	10ml storage buffer
   #A	8 x 140µl hybridization buffer
   #C	8 x 100µl wash buffer



3. [  ]  Amplification premix: add to 50ml tube with 12ml 5M Betaine:

   conc. stock 30ml 60ml Betaine 2M 5M 12ml / 12.88g 24ml / 25.75g / Cluster buffer 1x 10x 3ml 6ml H2O   mQ 15ml 30ml

   • vortex;
   • 5min 0°C (the solution should become clear);
   • filter through 0.2µm Steriflip unit;
   • transfer:
        #11 (ampl. premix): 12ml
        #1 (Bst. mix): 12ml
        #D (amplif. premix): 8 x 100µl
4. [  ]  prepare IEM (Initial Extension mix):

   Amplification premix	975µl
   10mM dNTP's	20µl
   Taq polymerase	5µl
   total	1ml

   • transfer 8 x 120µl in #E(extension mix);


5. [  ]  prepare Bst-amplification mix (tube #1):

   Amplification premix	12ml
   10mM dNTP's	240µl
   Bst polymerase	120µl
   total	12.36ml



6. [  ]  denaturized library:
   

   conc. stock library 1nM NaOH 0.1N 2N EB Qiagen

   • vortex, spin down, 5min RT,
   • prepare tubes with hybridization buffer (one tube per library) and transfer calculated volume (at least 2µl) of denaturized library in each;
   • vortex, spin down, transfer 120µl of template per channel #B (template),
   • keep strip #B on RT till loading on the FlowCell.


7. [  ]  take following things to CS:
   • hybridization manifold;
   • amplification manifold;
   • hybridisation buffer (for the case of problems with manifold);
   • 1ml and 200µl pipettes.

Cluster generation

1. turn on CS, wait 30 s, turn on computer;
2. empty the waste container, wipe the CS and table with a wet paper towel;
3. wash CS, ~10min:
   • change water in tubes: 1,9,10,11,12;
   • open "Amplification_only_v3.0" or "PE_Amplification_only_35cycles_v3.1" recipe;
   • select "WashAmponlyLines", "Start", "OK";
"User Wait" after the washing step & follow messages on the screen


4. [  ]   template loading:
   • remove H₂O from tubes in reagent positions 1,9,10,11,12 ⇒ OK
        prime air 1,9,10,11,12;
   • load reagents in positions 1,9,10,11,12 ⇒ Cancel
   • remove air from the washing bridge [26 · 40µl/min · 120µl];
   • remove washing bridge;
   • resume the recipe ⇒ OK
   • load FlowCell, tube strip holder, hybridization manifold, strip A (hybridization buffer) ⇒ OK
     CHECK!!! that all channels behave similarly,
        [60µl/min · 120µl];
   • strip #B(template) ⇒ OK (time for heating),
        96°C · 1°C/s
        [15µl/min · 75µl] 5 min;
        [100µl/min · 10µl];
        wait 30s
        40°C · 0.05°C/s (time for cooling)
   • strip #C(wash buffer) ⇒ OK,
        [15µl/min · 75µl] 5 min;
        40°C · 1°C/s
   • strip #D(amplif. premix) ⇒ OK,
        [15µl/min · 70µl] 5 min;
   • strip #E(extension mix) ⇒ OK,
        [60µl/min · 95µl] 1,5 min;
        74°C · 1°C/s
        wait 90s
        60°C · 1°C/s
   • strip #0(empty tube) ⇒ OK,
        [15µl/min · 20µl];


5. [  ]   install amplification manifold and check (IMPORTANT!) liquid flow in all channels:
   • remove hybridization manifold (put it on clean surface), connect amplification manifold ⇒ Cancel;
   • manually prime line 12 (storage buffer);
   • pump storage buffer and check liquid flow in all channels [12 ·60µl/min · 100µl];
   • pump small air gap [26 ·40µl/min · 10µl];


6. [  ]   cluster amplification:
   • resume the recipe ⇒ OK: "Check for proper flow through the FlowCell and manifold" ⇒ OK;
        prime reagents 1,9,10,11,12;
     

     35 cycles:

     #9(formamide) [30µl/min · 28µl]
     #11(ampl. premix) [30µl/min · 28µl]
     #1(ampl. mix) [30µl/min · 36µl]

     
        #10(wash buffer) [30µl/min · 140µl],
        20°C · 1°C/s,
        #12(storage buffer) [15µl/min · 95µl],
        20°C · 1°C/s;


7. there are two possibilities to proceed:
   • FlowCell storage (see below);
   • cluster check on fluorescent microscope.

FlowCell storage

1. air gap [26 · 30µl/min · 40µl];
2. unclip "reagent delivery port" & "central clamp" of amplification manifold;
3. remove FC and accurately clean it with water and lens cleaning tissue;
4. put FlowCell in 50ml tube with Storage buffer (original storage tube), store at +4°C;

CS wash

1. wash amplification and hybridization manifolds (see "Washing of manifolds");
2. lines washing should be perfomed with washing bridge attached;
3. pour fresh water in water-tubes of the used positions X = 1,9,10,11,12:
   • remove tube with reagent;
   • air gap [X · 240µl/min · 30µl], during the air pumping rinse the end of the tubing using washing bottle;
   • attach tube with water;
4. open "Amplification_only_v3.0" or "PE_Amplification_only_35cycles_v3.1" recipe;
5. select "WashAmponlyLines", "Start", "OK", ~10min;
6. close the program; turn off computer, turn off CS.

Notes

• volumes of reagents after reaction (per tube):

  position reagent initial volume should remain #A Hybr. buffer 140µl 20µl #B template 120µl 35µl #C Wash buffer 100µl 25µl #D Ampl.premix 100µl 30µl #E Extention mix 120µl 25µl #1 Bst mix 12.36ml ~4ml #9 formamide 15ml ~8ml #10 Wash buffer 10ml ~9ml #11 Ampl.premix 12ml ~5ml #12 Storage buffer 10ml ~9,5ml