Cluster control

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problems/questions
  • Solutions: SB preparation calculation (from seq.molbiol.ru)
  • new microscope: differences should be inserted


NB! post-PCR operation

Содержание


[править] Prepare reagents

in advance thaw 2 ml aliquote of staining buffer (SB);

  1. combine 2ml SB + 0.4µl SYBR Green I and mix;
  2. Label and fill in strips:
    • 2x #S: 8x 160 µl of HT1 (hybridization buffer),
    NB! do not prepare #S strips, if the FC is stained directly after amplification; HT1 (hybridization buffer) is in position 12
    • 2x #Y: 8x 120 µl of staining solution (for the control FC)
    • #W: 8x 240 µl of H2O,
    • #O: empty
  3. check you have:
    • staining manifold
    • control FC with clusters
    • 2x Petri dishes
    • small pieces of parafilm


[править] Flow Cell staining

  1. install staining manifold:
    after amplificationafter storage
    • air gap [26 · 40µl/min · 120µl]& remove amplification manifold;
    • install staining manifold;
    • turn on CS;
    • wait 30s, turn on computer;
    • check/empty waste container;
    • air gap [26 · 40µl/min · 120µl] & remove washing bridge;
    • install FC & staining manifold;
  2. install tube strip holder;
  3. staining:
    • strip #Y (staining solution)[26 · 30µl/min · 100µl];
    • strip #O (air gap), [26 · 40µl/min · 25µl];
    • unclip «reagent delivery port» & «central clamp» & rise up staining manifold;
    • remove FC;
  4. install control FC & attach the staining manifold back;
  5. repeat step 3 for control FC;
  6. install empty FC and attach the staining manifold back;
  7. prepare FCs for imaging:
    • clean FCs with water and lens cleaning tissue;
    • put FCs in Petri dishes, close holes with pieces of parafilm;
  8. take into microscope room:
    • FCs;
    • lens cleaning tissue;
    • wash bottle;
    • calculator;
    • pen;
    • this protocol


[править] Fluorescent microscope

  1. if necessary, turn on:
    • microscope power supply (Fig 1: 3);
    • FluoArc-lamp (Fig. 1: 2);
    • computer (login: Z1imager, password: zeiss);
    NB! if you turn off the lamp, you should wait at least 20min before to turn it on again
  2. install control FC:
    • set the lamp intensity: 20%(Fig 1: 1);
    • click «Load position» on the touchscreen (Fig. 2: 5);
    • clean the microscope table (Fig.1: 7) with optic cleaning pad (Fig. 1: 4);
    • set regulator position to the right (Fig. 2: 2) to see image on the screen;
    • select the 20x objective;
    • check, that channels of control FC are filled with staining solution;
    put the control FC to the «right-back» corner of the table (Fig. 3);
    NB! FC sits loosely in the standard glass holder
  3. start program:
    • program AxioVision Rel. 4.5;
    • open Microscope Window (MW) from the main menu: «Microscope» ⇒ «microscope»;
    • set coordinates: X=##, Y=##, Z=0 (in the bookmark «Stage» of the MW);
    • click on the icon «Live» in the main menu;
  4. find the clusters on the control FC:
    • select «trunsmitted» in the MW;
    • open the «transmitted light» shutter, find edge of the 8th channel (Fig.2: 3&4), close shutter;
    • select «reflected» in the MW (38HEgreen filter);
    • open shutter, adjust focus distance (Fig. 2: 4) to see clusters on the bottom, close shutter;
    NB! clusters are on both internal surfaces of the FC; the shift «top ⇒ bottom» is +75µm
    NB! use «Best Fit» and «Exposure time» buttons
  5. put control FC back in Petri dish;
  6. check clusters on test FC (reflected light):
    • check, that channels of test FC are filled with staining solution;
    • put the test FC on the microscope table;
    • open shutter, find bottom clusters in the center of 8th channel (Fig.2: 3&4), close shutter;
    • calculate Y-positions for all channels. Distance between middles of two neighbour channels is 1800µm
    channelCalculationY coordinate
    8 Y
    7 Y +1800
    6 Y +3600
    5 Y +5400
    4 Y +7200
    3 Y +9000
    2 Y +10800
    1 Y +12600
    • take pictures from all channels:
    NB! UV is bad for clusters, be quick
    • open shutter;
      • adjust the focus to see bottom clusters;
      • take the picture: «Snap» in the «Live» window;
      • close shutter;
      • save picture in the FC folder (format: solexa_2008_19_16_chX.tif);
      • change Y position in the MW to move to another channel;
    • finish:
      • click «Load position» on the touchscreen (Fig. 2: 5);
      • put the test FC back in Petri dish;
      • close the AxioVision program;
      • transfer files to your computer;
    • if you are the last user:
      • turn off the Power Supply (Fig. 1: 3);
      • turn off the FluoArc lamp (Fig. 1: 2);
      • logout.


[править] Flow Cell wash for further storage

  1. fill stained FCs with HT1 (hybridization buffer) solution:
    after storageafter amplification

    for each stained FC:

    • unclip «reagent delivery port» and «central clamp» of staining manifold;
    • remove empty FC;
    • install the stained FC & attach the staining manifold back;
    • strip #S (HT1 (hybridization buffer)) [26 · 40µl/min · 120µl];
    • strip #O (air) [26 · 40µl/min · 25µl];
    • unclip «reagent delivery port» and «central clamp» of staining manifold;
    • remove FC
    • remove staining manifold and empty FC;

    for each stained FC:

    • install stained FC and used amplification manifold;
    • HT1 (hybridization buffer): [12 · 40µl/min · 120µl];
    • air gap [26 · 40µl/min · 25µl];
    • unclip «reagent delivery port» and «central clamp» of staining manifold;
    • remove FC;

    wash amplification manifold:

    • install empty FC & attach amplification manifold back;
    • H2O [12 · 60µl/min · 240µl];
    • air [26 · 40µl/min · 120µl];
    • remove amplification manifold
  2. clean FC with water and lens cleaning tissue;
  3. put Flow Cell in 50ml tube with HT1 (hybridization buffer), store at 4°C;
  4. wash empty FC and staining manifold:
    • install empty FC & attach the staining manifold back;
    • strip #W (H2O) [26 · 60µl/min · 240µl];
    • strip #O (air) [26 · 40µl/min · 120µl];
    • remove empty FC & staining manifold;
  5. install bridge manifold;



[править] Flowcell processing for sequencing

  1. wash empty FC and staining manifold:
    • strip #W(H2O) [26 • 60µl/min • 240µl];
    • strip #0(air) [26 • 40µl/min • 120µl];
    • remove empty FC & staining manifold;
    • clean flowcell area with H2O and lens cleaning tissues;
  2. install «linearization/ blocking/ hybridization» reagents;
  3. fill control FC with storage solution:
    • install control FC & attach the staining manifold back;
    • storage solution [12 • 40µl/min • 120µl];
    • air gap [26 • 40µl/min • 25 µl];
    • unclip «reagent delivery port» and «central clamp» of staining manifold;
    • remove control FC clean it with water and lens cleaning tissue;
    • put control FC in 50ml tube with storage buffer, store at 4°C;
  4. install test FC;
  5. storage solution [12 • 40µl/min • 120µl];
  6. go to linearization/ blocking/ hybridization protocol;


[править] Solutions

Staining buffer (SB), store at −20°C

insert calculation

  • vortex, filter through 0,2µm filter;
  • make 2ml aliquots;
  • freeze in liquid nitrogen and store at −20°C

SYBR Green I

  • thaw stock solution to room temperature;
  • prepare 0.4µl aliquots in 0.5ml tubes;
  • freeze in liquid nitrogen and store at −20°C dessicated;


[править] Notes

Fluorescent microscope IMAGER.Z1 (Zeiss Axiomager.Z1)T1, Room 22

  • online booking: date and time
  • computer: Lab-RR/Mg04-33
  • responsible: Rudi Lurz; T1, R308; phone(+49 30)8413 1644 lurz@molgen.mpg.de
  • keys: abt. Hermann, Rudi Lurz, Bodo Lange, Sylvia Krobitsch
Fig. 1. 1 — lamp regulator; 2 — UV-lamp; 3 — microscope power supply; 4 — optic cleaning pads; 5 — optic cleaning sticks; 6 — image output selector; 7 — microscope table.


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