Prepare reagents

1. combine 2ml SB + 0.4µl SYBR Green I and mix;
2. label and fill in strips:
   NB! do not prepare #S strips, if the FC is stained directly after amplification; storage buffer is in position 12
   • 2x #S: 8x 160µl of storage buffer,
   • 2x #Y: 8x 120µl of staining solution (for the control FC)
   • #W: 8x 240µl of H₂O,
   • #O: empty
   
   


3. check you have:
   • staining manifold
   • control FC with clusters
   • 2x Petri dishes
   • small pieces of parafilm

Flowcell staining

1. install staining manifold:

   after amplification air gap [26 · 40µl/min · 120µl] & remove amplification manifold; install staining manifold;

   after storage 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 #0 (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;

Fluorescent microscope

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
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
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;
find the clusters on the control FC:
• select "transmitted" in the MW;
• open the "transmitted light" shutter, find edge of the 8^th 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
put control FC back in Petri dish;
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 the 8^th channel (Fig.2: 3&4), close shutter;
• calculate Y-positions for all channels:
       flowcell type:  old  new

  Channel Y-position 8 7 6 5 4 3 2 1



• 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_10_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.

Flowcell wash for storage

1. fill stained FCs with storage solution:

   after storage 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(storage solution) [26 · 40µl/min · 120µl]; strip #0(air) [26 · 40µl/min · 25 µl]; unclip "reagent delivery port" and "central clamp" of staining manifold; remove FC

   after amplification remove staining manifold and empty FC; for each stained FC: install stained FC and used amplification manifold; storage solution: [12 · 40µl/min · 120µl]; air gap [26 · 40µl/min · 25 µl]; 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 flowcell in 50ml tube with storage 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 #0(air) [26 · 40µl/min · 120µl];
   • remove empty FC & staining manifold;
5. install bridge manifold;

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;

Solutions

Staining buffer (SB), store at -20°C
conc. stock 10ml 50ml L(+) Ascorb. acid Na salt 0.1M 198.1g/M 0.2g 0.99g TrisCl, pH 8 100mM 1M 1ml 5ml H2O   mQ 4ml 45ml
• vortex, filter through 0,2µm filter;
• make 2ml aliquots;
• freeze in liquid nitrogen and store at -20°C


SYBR Green I
• heat stock solution to NT;
• prepare 0.4µl aliquotes in 0.5ml tubes;
• freeze in liquid nitrogen and store at -20°C dessicated;





Notes

Fluorescent microscope IMAGER.Z1 (Zeiss Axioimager.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. Herrmann, 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.





Fig. 2.  1 — microscope table; 2 — image output selector; 3 — XY-ajustment; 4 — Z-ajustment; 5 — touchscreen.





Fig. 3.  FC-position in the right corner of the slide holder.







Second-generation sequencing
URL: http://seq.zbio.net
e-mail: soldatov@molgen.mpg.de
visits:
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