• Home »
  • Next generation seq.
  • Terms
  • Applications
  • Schemes »
    • Fragment libr. prep
    • Mate-Paired libr. prep
    • Clonal amplification
    • Polymerization-based sequencing
    • Ligation-based sequencing
• Illumina »
  • Library »
    • Fragment
    • Mate-Paired
    • Mate-Paired Long
    • Library amplification
  • Cluster Station »
    • Cluster generation
    • Cluster control
    • FlowCell processing
    • Multiple primer hyb.
    • DECON wash
  • 2G analyzer »
    • Focusing
    • Reagents
    • Sequencing (first read)
    • Sequencing (PE read)
  • Data analysis
  • Illumina materials
• SOLiD »
  • Library »
    • Fragment
    • Mate-Paired
    • Mate-Paired Long
    • Library amplification
  • ePCR »
    • ePCR amplification
    • Bead wash
    • Bead enrichment
    • 3'-end modification
  • Sequencing »
    • Bead deposition
    • Flowcell installation
    • QC-run
    • Sequencing run
    • Enzyme and Primer plates
  • SOLiD Kits handling
  • SOLiD materials
• General »
  • Solutions
  • Consumables
  • Kits and enzymes
  • DNA »
    • RNase treatment
    • Shearing
    • Purification
  • RNA »
    • mRNA purification on Dynabeads
    • ds cDNA synthesis
  • Oligonucleotides
  • NA quantitation
  • NA electrophoresis
  • Agarose
  • PAAG
  • Data analysis »
    • UNIX comands

QC-run

Panel Position Map Notes  Comments

NB! post-PCR operation. This protocol is for FRAGMENT library / M-P libraryM-P library / FRAGMENT library

1. for each library deposit 15x10⁶ beads on 4-well slide;



2. check, that Instrument Buffers are installed, mark buffer levels:
   

   Reagent live time [days] volume fo QC run [ml] Cleave Solution 1 5 ---- Reset Buffer 5 30 Cleave Solution 1 5 ---- 1x Instrument buffer 10 70 Storage buffer 10 20




3. to create QC-run:
   • click "Create Run …";
   • select "Titration/QC Run" & Next;
   • (optional) change name, insert comments & Next;
   • select position of each sample (orientation as for closed FlowCell) & Finish;
   • (optional) repeat procedure for second FlowCell;



4. prepare & install 48-well enzyme plate:
   • shortly vortex/spin-down T4 DNA ligase and put it in -20°C cold-box;
   • pipette 10µl of T4 DNA ligase in A1 (for flowcell 1, left) and A5 (for flowcell 2, right);

   ⁄ 1 2 3 4 5 6 7 8 A T4       T4       B                 C                 D                 E                 F                   flowcell 1 flowcell 2

   • seal plate;
   • keep at -20°C until use;



5. prepare and install Probe/Primer plate:
   • (i) melt, (ii) vortex, (iii) spin-down and (iv) put in ice-box:
       •   5x T4 DNA ligase buffer: 90µl/FC;
       •   mixA: 24µl/FC;
       •   1A: 15µl/FC;
       •   FM1: 2µl/FC;
       •   FM2: 10µl/FC;
       •   P2B: 10µl/FC;
       •   IA1, IA2: 10µl/FC each;
   
   
   
   • on NT:
       •   H₂O: 1.2ml/FC;
       •   Sodium Acetate: 20µl/FC;
       •   20X SSPE: 46µl/FC;
   
   
   
   • prepare and label: (1,7ml normal tubes):
     ◊   #LP (Ligation Probe Mixture)

     1 FC 2 FC H2O 306µl 612µl 5x T4 DNA ligase buffer 90µl 180µl Sodium Acetate Solution 20µl 40µl mixA (Probe Mix A) 24µl 48µl Total: 440µl 880µl

     
     
     
     ◊   #PO (Priming Oligo Mixture) for Fragment library

     1 FC 2 FC H2O 382µl 764µl 20X SSPE Buffer 23µl 46µl Tag 1 Seq Primer A 15µl 30µl P2B (P2 Block, 200 µM) 10µl 20µl Total: 450µl 900µl

     ◊   #PO (Priming Oligo Mixture) for M-P library

     1 FC 2 FC H2O 402µl 804µl 20X SSPE Buffer 23µl 46µl Tag 1 Seq Primer A 15µl 30µl P2B (P2 Block, 200 µM) 10µl 20µl IA1 (IA Block 1, 200 µM) 10µl 20µl IA2 (IA Block 2, 200 µM) 10µl 20µl Total: 450µl 900µl

     
     
     
     ◊   #PS (Pre-scan Reagent Mixture)

     1 FC 2 FC H2O 415µl 830µl 20X SSPE Buffer 23µl 46µl FM1 (Focal Map P1 Label, 200 µM) 2µl 4µl FM2 (Focal Map P2 Label, 200 µM) 10µl 20µl Total: 450µl 900µl

     
     
6. prepare plate:
   • put reagent into positions: (H1: LP), (H11: PS), (H12: PO);
   • seal plate;
   • if it will be used within 4 days, keep plate at 4°C, otherwise, snap freez in liquid nitrogen and keep at -20°C;

   1 2 3 4 5 6 7 8 9 10 11 12 A                         B                         C                         D                         E                         F                         G                         \ H LP                   PS PO




7. run:
   • install QC-FlowCell;
   • click "Start Run";
   • when finished: Tools > Generate Titration/QC Report (take ~10 min);



8. analyzis:

   parameter         P2/P1 ratio         on-axis beads         titration metric         P2 average

   • recalculate the proper P2 concentration of beads:
     c  =  (P2 average) x 495 / (QC-volume)

Panel Position Map

1 96 97 98 99 100 95 94 93 92 91 86 87 88 89 90 85 84 83 82 81 76 77 78 79 80    2 71 72 73 74 75 70 69 68 67 66 61 62 63 64 65 60 59 58 57 56 51 52 53 54 55    3 46 47 48 49 50 45 44 43 42 41 36 37 38 39 40 35 34 33 32 31 26 27 28 29 30    4 21 22 23 24 25 20 19 18 17 16 11 12 13 14 15 10 9 8 7 6 1 2 3 4 5

Notes

• Workflow:

  stage time prepare 4-wells dep. chambers in advance bead deposition ~3h set-up run ~10min buffers & waste ~15min enzyme & buffer plates ~0.5h install slide ~20min run 3h report & calculate dep. volumes ~10min