Preliminary

• book the Cluster Station (CS);
• prepare Cluster Generation Kit Box 1 (-20°C) and Box 2 (NT);
• prepare PE Cluster Generation Kit Box 1 (-20°C) and Box 3 (NT);
• thaw:
  • 10X Linearization 1 Buffer;
  • 10X Blocking Buffer;
  • 2.5mM ddNTP Mix;
  • TrisCl, 1M;
  • Primer v2;
  • Rd1 PE Seq Primer;
• put Terminal Transferase into -20°C box;
• put into -20°C box:
  • linearization 1 Enzyme;
  • Blocking Enzyme A;
  • Blocking Enzyme B;

Prepare reagents

NB! store all components on ice

1. label tubes and store them on ice:
   • 15ml tubes:

     #3	linearization mix

   • 5ml tubes:

     #LB	1x Linearization Buffer
     #BB	1x Blocking Buffer

   • 1.7ml screw-cap tubes:

     #5	blocking mix
     #7	sequencing primer mix
     #14	H2OLinearization 1 Mix
     #15	blocking buffer
     #16	1x Linearization Buffer
     #17	0.1N NaOH (from the kit)
     #18	1x TE (from the kit)

   • 2ml screw-cap tubes:

     #6	1x Blocking Buffer
     #8	Blocking Mix

   • 50ml screw-cap tubes:

     #10	wash buffer
     #12	storage buffer



2. fill tubes:

   #10	wash buffer, 515ml
   #12	storage buffer, 510ml
   #14	H2O, 1.5ml / 2x750µl



3. prepare kits:
   • buffers: vortex briefly and spin down;
   • enzymes: spin down, mix by finger tipping, spin down again & put in -20°C box;


4. prepare:
   • 1x linearization buffer in 5ml tube #LB:

     3ml 10x linearisation 1 buffer 300µl H2O 2.7ml / 3x 900µl

     put 1.3ml / 2x650µl into tube #16;
     
     
   • 1x blocking buffer in 5ml tube #BB:

     5ml 10x blocking 1 buffer 500µl H2O 4.5ml / 5x900µl

     put 2ml into tube #6;
     
     
   • linearisation 1 mix in tube #14:

     1.3ml 1x linearisation buffer 1.29ml / 2x643.5µl linearization 1 Enzyme 13µl

     
     
   • blocking mix in tube #8:

     1.3ml 1x blocking buffer 1.53ml / 2x764.5µl ddNTP, 2.5mM 67µl Blocking Enzyme A 20µl Blocking Enzyme B 84µl

     
     
   • Na Periodate solution:
     Na Periodate + 1518µl H₂O / 2x759µl & vortex;
     
     
   • linearization mix in tube #3:

     3ml Formamide 1.5ml TrisCl, pH8, 1M 60µl --- mix --- Na Periodate solution 1.44ml / 2x718.5µl --- mix --- APL 2.3µl --- mix ---

     
     
   • 1x blocking buffer in tube #15:

     1.4ml 10x blocking 1 buffer 140µl H2O 1260µl / 2x630µl

     
     
   • blocking mix in tube #5:

     1.56ml H2O 1.36ml / 2x679.5µl blocking buffer, 10x 151µl ddNTP, 130µM 30µl --- mix --- TdT 20µl

     
     
   • sequencing primer mix in tube #7:

     1.3ml hybridization buffer 1.31ml / 2x657µl sequencing primerRd 1 PE Seq Primer 6.6µl

   
   


5. 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;

Linearization, blocking and 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. wash CS, ~1015min:
   • change water in tubes: 3,5,7,10,12,14,15,17,186,7,8,10,12,14,16,17,18;
   • open "Linearization_Blocking_Primerhyb_v3.0PE_2P_R1prep_Linearisation_CombinedBlocking_PrimerHyb_v2.0" recipe;
   • select "WashLinearizationBlockingPrimerhybLinesWashREAD1LinearizationandCombinedBlockingprimerhybLines", "Start", "OK";


"User Wait" after the washing step & follow messages on the screen


4. load reagents:
   • remove H₂O from tubes in reagent positions 3,5,7,10,12,14,15,17,186,7,8,10,12,14,16,17,18 ⇒ OK
        prime air 3,5,7,10,12,14,15,17,186,7,8,10,12,14,16,17,18;
   • load reagents in positions 3,5,7,10,12,14,15,17,186,7,8,10,12,14,16,17,18 ⇒ Cancel;


5. load FC:
   • remove liquid from the washing bridge [26 · 40µl/min · 120µl];
   • remove washing bridge;
   • install flowcell and amplification manifold;
   • manually prime #12 (storage buffer);
   • pump storage buffer and check the proper flow through all channels [12 · 60µl/min · 100µl];
   • pump a small air gap [26 · 40µl/min · 10µl];


6. linearisationread 1 processing:
   • resume the recipe ⇒ OK
     prime reagents 10,14,16,12
        20°C · 1°C/s
     [16 · 15µl/min · 95µl]
     [14 · 15µl/min · 95µl]
        37.9°C · 1°C/s
        wait 30 min
        20°C · 1°C/s
     [10 · 15µl/min · 95µl]
     [12 · 15µl/min · 95µl]
     
   • combined blocking 1:
     prime reagents 8,6
        20°C · 1°C/s
     [6 · 15µl/min · 90µl]
        38°C · 1°C/s
     [8 · 15µl/min · 95µl]
     9x: [8 · 15µl/min · 5µl]
     
   • combined blocking 2:
        60°C · 1°C/s
     [8 · 15µl/min · 25µl]
        wait 15 min
     
   • combined blocking wash:
        20°C · 1°C/s
     [10 · 15µl/min · 95µl]
     [12 · 15µl/min · 95µl]
     
   • denaturation & hybridization:
     prime reagents 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]
     
   • resume the recipe ⇒ OK
     prime reagents 3,14
        20°C · 1°C/s
     [3 · 15µl/min · 300µl]
     [14 · 15µl/min · 95µl]
     
   • blocking:
     prime reagents 10,5,15
        20°C · 1°C/s
     [15 · 15µl/min · 90µl]
        38°C · 1°C/s
     [5 · 15µl/min · 95µl]
     9x [5 · 15µl/min · 5µl]
        20°C · 1°C/s
     [10 · 15µl/min · 95µl]
     
   • primer hybridisation:
     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. remove FC:
   • air gap [26 · 15µl/min · 40µl];
   • unclip "reagent delivery port" & "central clamp" of amplification manifold;
   • remove FC and accurately clean it with water and lens cleaning tissue;
   • transfer flowcell to Genome Analyser;
   • install empty FC and attach the amplification manifold back;


8. CS post-wash:
   • wash amplification manifold (see "Washing of manifolds");
   • install washing bridge;
   • pour fresh water in water-tubes of the used positions X = 3,5,7,10,12,14,15,17,186,7,8,10,12,14,16,17,18:
     • 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;
   • open "Linearization_Blocking_Primerhyb_v3.0PE_2P_R1prep_Linearisation_CombinedBlocking_PrimerHyb_v2.0.xml" recipe;
   • select "WashLinearizationBlockingPrimerhybLinesWashREAD1LinearizationandCombinedBlockingprimerhybLines", "Start", "OK", ~15min;
9. close the program; turn off computer, turn off CS.

Notes

• If only linearization and blocking are performed, prepare reagents for positions: 3,5,10,12,14,15 and use the "Linearization_Blocking_only_v3.0" recipe
  
  
• Unlike to single-read linearization-blocking-primer_hyb protocol it is not recommended to store the FC after blocking. So, if multiple primer hybridisation has to be performed do it immediately after the blocking step ("Primerhyb with multiple primers" protocol).
  
  
• Volumes of reagents after reaction:

  position reagent initial volume should remain #6 1x Block. buffer 2ml ~1ml #7 seq. primer mix 1.32ml ~300µl #8 Blocking mix 1.7ml ~150µl #10 Wash buffer 15ml ~12.4ml #12 Storage buffer 10ml ~7.4ml #14 Linearisation 1 mix 1.3ml ~230µl #16 1x Linear. buffer 1.3ml ~230µl #17 NaOH, 0.1M 1.5ml ~430µl #18 1x TE 1.5ml ~430µl

  position reagent initial volume should remain #3 Linearisation mix 3ml ~600µl #5 Blocking mix 1.5ml ~710µl #7 Seq. primer v2 1.3ml ~620µl #10 Wash buffer 5ml ~3.4ml #12 Storage buffer 5ml ~4.2ml #14 H2O 1.5ml ~700µl #15 Blocking buffer 1.4ml ~680µl #17 NaOH, 0.1M 1.5ml ~800µl #18 1x TE 1.5ml ~800µl