Sequencing (first read)

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problems/questions

 Problem list:

  • how much free space on drive D is required for the run (maybe a table: type of run / disk space)


Содержание


[править] Preliminary

    • check the disk space (should be ### on drive D);
    • wash the GA and the table with a wet paper towel;
    • perform the GA pre-run wash;
    • prepare


[править] Loading reagents

  1. load reagents in the order:
    1 IMR
    3 SMX
    2 PW1
    4 PR1
    5 PR2
    7 PR3
    6 CLM
  2. perform reagent priming:
    • place the bundled tube ends in the 15ml tube;
    • open "GA2_Prime_v8" recipe ⇒ Start
    [X · 250 · 2500 · 100] takes ~5min
    • check the waste volume (should be ~6.4ml) and record it in the run list;
    • place the bundled tubing back to the waste bottle


[править] FC installation

    NB! use only lens cleaning tissue; fold it to use the clean, untouched part each time
  1. remove the old FC:
    • open "Manual Control/ Setup" ⇒ "Load Flow Cell";
    • "Instrument" ⇒ "Unlock door";
    • pump: [to: Flow Cell • solution: 28 • volume: 0 • asp.rate: 250 • disp.rate: 2500] ⇒ Enter;
    • lift manifolds;
    • remove the old FC, blot oil with lens cleaning tissue, wash FC with ethanol, put back into the original tube;
    • lift the beam dump;
    • remove the prism, put it on a piece of lens cleaning tissue;
  2. clean the imaging compartment to remove dust and oil with lens cleaning tissue damped with ethanol: Peltier element, FC table, reachable metal surfaces;
  3. manifolds cleaning:
    • clean manifolds with lens cleaning tissue damped with water;
    • blot with a dry lens cleaning tissue;
    • clean manifolds with lens cleaning tissue damped with ethanol (only metal parts!);
    • blot with a dry lens cleaning tissue;
  4. gently wash prism and prism metal base with a stream of ethanol from a washing bottle and blot with lens cleaning tissue;
  5. change gloves;
  6. prepare prism:
    • ○○○   3x clean prism with lens cleaning tissue damped with "clean ethanol" (from 2ml aliquotes):
    » wipe prism sides with a single motion (should be a distinct sound);
    » refold lens cleaning tissue;
    • ○○   2x clean prism with dry lens cleaning tissue:
    » wipe prism sides with a single motion;
    » refold lens cleaning tissue;
    • check washing quality on reflected light, repeat cleaning if necessary;
    • install the prism in the imaging compartment;
  7. clean FC:
    • blot FC using lens cleaning tissue;
    • clean both sides with lens cleaning tissue wetted in water;
    • change gloves;
    • ○○○   3x clean both sides with lens cleaning tissue damped with "clean ethanol" (from 2ml aliquotes):
    » wipe of prism sides with a single motion (should be a distinct sound);
    » refold lens cleaning tissue;
    • ○○   2x clean both sides with dry lens cleaning tissue:
    » wipe of prism sides with a single motion;
    » refold lens cleaning tissue;
    • check washing quality on reflected light, repeat cleaning if necessary;
  8. install the FC:
    • move FC to the extreme right and forward position;
    • hold the FC & slowly lower the manifolds;
    • press manifolds downward to ensure the proper contact;
  9. check reagent delivery by pumping PR2 (5) through the FC:
    • place the bundle of the waste tubings in a 1.5ml tube;
    • pump: [to: flowcell • solution: 5 • volume: 100 • asp. rate: 250 • disp. rate: 2500] ⇒ Enter;
    • check for bubbles: if bubbles appear in the inlet manifold — reinstall the FC;
    • check the volume of the waste: should be 750-800µl;
    • repeat the reagent delivery check two more times: record delivered volumes into the run list;
  10. applying oil:
    • pipette about 140µl of oil, wipe the tip from outside with lens cleaning tissue;
    • dispense the oil slowly between the FC and prism, pressing the tip against the prism as far as possible from the edge of the FC. The oil front should move with a constant speed (if not — the FC was not installed correctly and should be reinstalled);
    • check with a folded lens cleaning tissue that the oil has reached the top, middle and lower right edge of the FC;
    • check that the top of the FC is clean and there are no bubbles between the prism and FC (if necessary, remove them by lens cleaning tissue or reinstall the prism and FC);
    • wipe the surface of the Peltier element with a lens cleaning tissue damped with ethanol;
    • close the door;
  11. run:
    • put waste tubing in the large waste bottle;
    • open the "Run" thumb;
    • open "GA2-151_SR_v8.3" ⇒ Start ⇒ OK;
    open "GA2-PEM_2x51_PE_v8.3" ⇒ Start ⇒ OK;
    first base incorpoaration and autofocusing: ~20min
    • open "View" ⇒ "Calibration results": goodness of fit ≥ 0.99; sensitivity 350-400
    • click "Accept" to accept the focus calibration. If necessary, move to another tile and repeat the autocalibration
    • open the the First Base Report and check the intensity values
    • click "OK" to resume the run
    51 cycles take ~2,5days
  12. second read
  13. after the run
    • perform the post-run wash
    • weigh reagent bottles and record results


[править] Notes

    • the diameter of the laser beam is about the same size as the size of the picture, so the quality of the optical surfaces should be extremely high — check using reflected light;
    • contact clean FC and prism only with clean (!) gloves or with a lens cleaning tissue. Do not rub surfaces, clean by blotting or one-way movements;
    • fold lens cleaning tissue accurately and use new clean area for every wipe;
    • channels of the FC should not be dried out: contact holes with lens cleaning tissue VERY ACCURATELY (especially if FC is oriented vertically). If some channel will dry out, put a drop of High Salt Buffer on the hole with a 200µl pipette — channel will be filled by capillary force;
    • leakage: GAII works under negative pressure. There should be no leakage except for one during connection/disconnection of tubing. The problem associated with leakage — if the FC or FC-manifold surface is dirty, the focal plane of the glass would be disturbed — result with uneven light, bad focusing.
    • bubbles disturb the focusing. Sign of problems: bubbles in the waste tubes. To localize the problem open the instrument door, pump scanning buffer through the FC (pump: [to: flowcell • solution: 3 • volume: 100 • asp. rate: 250 • disp. rate: 2500] ⇒ Enter), and try to localize in what part of the system bubbles appear. If bubbles appear in the FC input, slightly relax manifold and accurately push the flowcell in the correct position (upper-right).


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