Sequencing run

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NB! post-PCR operation


[править] Prepare in advance

  • check that data collected from the previous run are satisfactory;
  • empty the waste canister;
  • prepare:old
" two glass beakers for washing tubing;
" three glass beakers for O-ring wash:
" aspiration pump;
" waste bin for excess of 1x Instrument Buffer;
" 2ml tubes for control of deposition;
" screwdrivers for bolts and for deposition chamber;
" place, where to put the old assembly (storage chamber or PP-container for slides);
" plastic box with water for plastic parts of deposition chambers;
" plastic box for metal parts of deposition chambers



  • prepare:new
"open Instrument Control Software(ICS)
"System Status menu => set the Chiller to «Cooling», (if it is not already) =>solutions can be installed if temperature is below 10°C
"check lamp hours and if it is necessary install a new one
"check that lamp is on (at least 3 hours before detect focus range)
"thaw : universal buffer part 1 and 1x T4ligase buffer part 1
"thaw the chemistry stripes you want to use in 4°C fridge in a 96 square- well storage plate
"cool the centrifuge to 4°C (after deposition)
"prepare buffers:
  1. 1xInstrument buffer
    • prepare Instrument buffer, store at 4°C for 10 days
    density1L
    H2O1g/ml700ml
    Instrument buffer, 10x1.014g/ml100ml / 101.4g /
    Glycerol1.262g/ml200ml / 252.4g /


  2. 1x T4 Ligase Buffer
    • transfer the contents of the 1xT4 Ligase buffer Part 1 to the 1x T4 Ligase buffer Part 2 bottle
    • mix the contents by slowly inverting the bottle to ensure mixing and to minimize bubbles
  3. Imaging buffer
    • transfer the contents of the Imaging buffer Part 1 to the Imaging buffer Part 2 bottle
    • mix the contents by slowly inverting the bottle to ensure mixing and to minimize bubbles
  4. Universal buffer
    • transfer the contents of the Universal buffer Part 1(stored at −20°C) to the Universal buffer Part 2 bottle
    • mix the contents by slowly inverting the bottle to ensure mixing and to minimize bubbles

    • apply the Universal Buffer label supplied with the bottle

  5. Cleave solution 2.1
    • transfer the contents of the Cleave Solution 2.1 Part 1(stored at −20°C) to the Cleave Solution 2.1 Part 2 bottle
    • mix the contents by slowly inverting the bottle to ensure mixing and to minimize bubbles

    [править] Installation

    1. install 1x Instrument buffer ,Storage buffer and the waste canister into position in the cabinet (waste canister should be empty or not more than ¼ full)
    2. install Cleave Solution 1,Reset buffer and Cleave Solution 2.1 into positions on the side of the instrument (check if there is enough volume of Reset buffer)
    3. install Imaging buffer, 1x T4 Ligase buffer and Universal buffer into positions in the chiller block
    4. prime the instrument and storage buffer lines (System Status => click «Prime») maybe it is necessary to do it twice, confirm that the buffer lines are filled
    5. prepare instrument flowcell:
    6. if the slides from the previous run is still inside

      • ensure that the run, images, and data collected from the previous run are satisfactory
      • in ICS: Clear Flowcell and Yes
      • open FlowCell chamber
      • remove old slide assembly and put slide in a chamber filled with Slide Storage buffer (work fast to prevent drying of slides)
      • remove O-Rings
      • clean the flowcell block with Kimwipes (first water and after that with 70% Ethanol=> carefully remove all liquid)
      • THOROUGHLY check integrity of O-ring
      • wash O-ring sequentially by water, 70% ethanol and water again
      • insert a clean O-ring into the groove on the flowcell
      • check evenness of O-ring by finger, re-install it if necessary
    7. open a new slide:
      • remove adhesive paper from portholes;
      • add 1x Instrument Buffer to the reservoir of deposition chamber;
      • with 1ml pipette aspirate SLOWLY the Deposition Buffer from the well and optionally put it into prepared 2ml tube (later control the absence of beads on magnetic stand);
      • diagonally loosen screws;
      • open assembly;
      • put additional 1x Instrument Buffer on the top of the slide;
    8. install slide:
      • find orientation of the slide carrier;
      • QUICKLY pour off the excess of the 1x Instrument Buffer and install slide carrier (work fast to prevent drying of slides);
      • gradually tighten two bolts;
      • check, that liquid does not move when tightening;
      • aspirate the excess of liquid from the Flowcell holder;
      • wipe first with wet and then with dry paper towel the glass surface;
      • rotate Flowcell up and lock;
      • click «Load Flowcell» button; click OK
      • wait, until Flowcell is loaded and open Flowcell down;
      • check, that there are not to much bubbles (otherwise repeat «Load Flowcell» command);
      • aspirate excess of the liquid from Flowcell block, liquid trap and clean it with 70% ethanol;
    9. clean glass slide:
      • 2x one-way wiping with lens-cleaning tissue slightly moistened with water;
      • 3x one-way wiping with lens-cleaning tissue slightly moistened with ethanol;
      • rotate Flowcell up and lock;
      • repeat «Load Flowcell» command and check for leaking
    10. install reagent stripes on the instrument
      • clean needle and bottom of the needle holder with Kimwipes
      • centrifuge the thawed reagent strips in 96-well storage plate at 160xg; 2 min.; 4°C
      • place the reagents in the positions in the chiller block

      (flowcell 1- front block; flowcell 2- rear block)

      • fix the cover over the reagent stripes with cover fasteners
    11. Create run:
      • click «Create Run …»;
      • select Type of run (Fragment , Mate-pair, Paired End , WFA or multiplexing)
      • enter run name, select run protocol (SOLiD 4 should be selected)
      • define primer set 1+ 2 and its read length
      • select appropriate mask => next
      • specify samples and analysis
      • select position of each sample (orientation as for closed Flowcell) and Next;
      • check all parameters (reference human: 1000genomes) and Finish
      • assign to the selected flowcell

      [править] Detect Focus Range

      • restart the ICS software before detecting the focus range
      • find the old «focus range» in the file C:/Documents and Settings/All Users/Application Data/Applied Biosystems/discovery/imager_local settings (link on the desktop):

      old focus: ____________ ____________ ____________ ____________

      • lock doors of the instrument;
      • menu: Window ►; Imaging System;
      • optional: have a look on beads before detecting the focus range to check bead density and if the beads are clumped
        • click Show flowcell ► enter number of flowcell
        • menu ►Stage template ► find .STG file of the run
        • move green-box cursor into upper-left corner
        • Set «filter status» to «WL» and «Nosepiece Status» set to «2»
        • Enter in filter status the old focus values and press go to
        • If imager is ready press acquire and auto exposure
        • Press go live
        • if the grey box in focus status is «active» move the focal distance by holding down Ctrl key and pressing the right or left arrow key
      • select Tools ► Detect Focussing Range
      • specify the stage template file by click on Browse ► select the file imagingMap.STG of the run you have created
      • select flowcell (using the drop-down menu) and click Go*when imager is done a dialog with new values appears; click Yes to replace the values in the local settings file (ensure that in imager local settings)
      • check the images at random locations to ensure that the beads are in focus
      • if both flowcells are running repeat the procedure now for the second flowcell

      NB! reinstall the glass if the «MAX-MIN» range exceeds 5000

      [править] Start the Run

      • Optional: close all windows and restart the ICS
      • Click « Start run», if you start both flowcells leave some seconds between starting the run on the second flowcell

      [править] Notes

      • Never scrape surfaces with paper towels. To remove liquid: press paper towel, wait few seconds and remove.
Источник — «http://molbiol.ru/wiki/Sequencing_run»


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