Cluster Station

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Cluster station 1 — reagent delivery port; 2 — flowcell area; 3 — waste bottle; 4 — central clamp; 5 — waste port.

Содержание


[править] Work area

  • clean work:
    • powder-free gloves
    • lens cleaning tissue
    • 1L bottle with mQ water
    • washing bottle with mQ water
    • 100ml glass beaker for rinsing tubes

  • boxes:
    • for storage of tube caps
    • for Washing bridge storage

  • instruments:
    • plastic tweezers
    • "empty" flow cell for washing of used manifolds
    • 1ml and 200µl tips
    • tubes: 50ml, 15ml, 2ml, 8-strips
    • tube holders: 50ml, 2ml

  • cluster staining:
    • staining manifold
    • Petri dishes for stained FCs
    • pieces of parafilm for stained FCs

  • writing:
    • marker
    • pen




[править] Flow cell (FC)

  • touch only with clean(!) gloves or with a lens cleaning tissue;
  • channels should not be dried out: contact WET holes with lens cleaning tissue VERY ACCURATELY!!! Especially if FC is oriented vertically;</li>
  • leakage: both CS and GA work under the negative pressure. So, there should be no leakage except for one during connection/disconnection of tubing. Real problems: blockage of one of the channels or bubbles in the line.




[править] Manifolds

  • tubings ~2.85µl/cm
    • amplification and hybridization manifolds: 2x [13cm, 37µl]
    • washing bridge [30cm, 86µl]

  • flowrate restrictions:
    • washing bridge: < 240µl/min
    • flowcell: < 60µl/min
    • to avoid small droplets in tubing: < 30µl/min

  • Priming lines:
    • 20µl are pumped in one priming step
    • 1-4, 9-18: 17 cycles
    • 5-8: 13 cycles




[править] Washing of manifolds

used manifolds should be washed and stored dry
  1. if necessary, remove the Flow Cell:
    • air gap
    manifoldprogram
    amplification26 · 30µl/min · 40µl
    hybridization26 · 30µl/min · 25µl
    washing bridge26 · 30µl/min · 120µl
    • unclip reagent delivery port and central clamp
    • raise up manifold
    • remove Flow Cell
  2. insert an empty Flow Cell and attach the manifold;
  3. wash with water:
    • amplification manifold: [x · 60µl/min · 200µl], x is a position of H2O tube
    • hybridisation/staining manifold: [26 · 60µl/min · 200µl] from a 8-strip with 240µl H2O per tube
  4. air gap:
    • amplification manifold: [26 · 40µl/min · 120µl]
    • hybridisation/staining manifold: [26 · 40µl/min · 120µl] from empty 8-strip
  5. unclip the manifold;
  6. clean the rubber parts with (i) wet and (ii) dry lens cleaning tissue;
  7. put manifold into container;
  8. write date on the container




[править] Unloading of manifolds

  1. air gap:
    manifoldprogram
    amplification26 · 30µl/min · 40µl
    hybridization26 · 30µl/min · 25µl
    washing bridge26 · 30µl/min · 120µl
  2. unclip from "left" to "right":
    • reagent delivery port
    • central clamp
    • waste port
  3. remove Flow Cell if necessary;
  4. wipe the ports and the flow cell stage with water and dry with lens cleaning tissue




[править] Maintenance of CS


  • after each usage:
» wash (i) used tubing and (ii) manifolds with H2O
» empty the waste bottle


  • before each usage:
» clean flowcell area with H2O and dry by lens cleaning tissues
» if CS was previously used by:
our groupchange water in water storage tubes (WST)
another groupreplace WSTs with tubing washing and air priming
another group (DECON wash)50ml and 15ml tubes
shake the old WSTs gently to wash the cap,
change water in WSTs and shake again,
remove drops of water from the caps by 1ml pipette,
replace WSTs with tubing washing and air priming


2ml tubes:

wash caps twice with water,
replace WSTs with tubing washing and air priming
Источник — «http://molbiol.ru/wiki/Cluster_Station»


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