CBot general information

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cBot

A Thermal Stage—The thermal stage holds the flow cell and controls its temperature throughout the run.
B Reagent Stage—The reagent stage holds the 96-well reagent plate, the templates, and any custom or specialty primers.
C Waste Bottle Compartment—The waste bottle compartment holds a sensorcontrolled 250 ml waste bottle that collects reagents after they have passed through the flow cell and manifold.
D Barcode Scanner—The on-instrument barcode scanner records the unique ID of the reagent plate and flow cell used with each run. The cBot uses this information to verify the compatibility of a selected protocol and reagents.
E Touch Screen Monitor—The touch screen monitor displays the software interface, which guides you through each step of the cluster generation process.



Flow Cell
The flow cell (FC) is a silica slide with eight lengthwise lanes

Size of the HiSeq FC v1: 25mm wide X 75mm long;
Lanes: 1.7 mm wide


All surfaces of the lanes are covered with a dense lawn of oligonucleotides P5 and P7, corresponding to the common flanking regions of sequencing libraries. Single read FC: P5 contains a cleavable group PE FC: both P5 and P7 contain cleavable groups



Operation times to be done




What is happening on the cBot?

  1. Loading of sequencing libraries onto the FC
    • hand step: libraries are diluted and denatured in alkali solution
    • libraries are pumped into the lanes of the flow cell
    • library molecules (templates) are captured by the oligonucleotides attached to the surface of the FC
  2. Primer extension
    • oligonucleotides bound to the library molecules are 3’-extended; now strand complementary to templates are covalently bound to the FC
    • the double-stranded molecule is denatured, and the original template is washed away
  3. Bridge formation and template amplification
    • free ends of the bound templates hybridize to adjacent lawn primers forming bridges
    • primers are extended resulting in a double-stranded DNA bridge.
    • The dsDNA is denatured, strands form new bridges, primers are extended — this process of iso-thermal bridge amplification is repeated 35 times
    The result of the bridge amplification is a cluster of ~ 2000 double-stranded bridges, both strands covalently bound to the FC by 5' ends.
  4. Double stranded amplification products are made single-stranded
    • P5 sequences are cut at the cleavable site
    • strands attached to the surface by P5 ends are washed off after denaturation
    • molecules in the clusters are now single-stranded
  5. Blocking of free 3' ends
    • free 3’-OH ends of the library molecules and of lawn-primers are blocked to prevent non-specific priming during sequencing
  6. Hybridization of sequencing primer
    • the FC with the synthesized clonal clusters is now ready to be sequenced on the HiSeq2000

* picture of cluster formation process should be inserted


Working place near cBot

  • thin and thick marker
  • pen
  • empty FC
  • washing bottle with mQ
  • dust-free paper towels
  • cBot Manifold for HiSeq FC
  • plastic forceps
  • dust-free





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