Bead deposition, SOLiD 3+

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NB! post-PCR operation

[править] Prepare in advance

notched side is the bottom of the slide

[править] Deposition chambers


  • NB! for SOLiD 3+ use Solid Opti Slide Carrier (marked with «OPTI» on the top and with a blue side);
  • check Slide- carrier and keep preselected pairs of « top and bottom» of Deposition chamber
  • clean the selected deposition chambers and slide carriers[1]
  • preheat at 37°C for several hours (on the metal floor of the 37°C incubator)
  • check the Deposition volumes of the Deposition chamber[2] with a common glass slide and mQ-water; note the volume


Deposition chamber                                                                          
Deposition volume (VD) in µl  


[править] Equipment and consumables

Kits required:

  • SOLiD™ Slide Kit — 4 x 2 Sequencing Slides, #4406813 — slides (stored in exsiccator)
  • SOLiD™ Bead Deposition Kit 2 of 3, #4392169 — deposition buffer
  • Optional: SOLiD™ Slide Kit — Slide Storage Buffer, # 4389772 — storage buffer

prepare:

  • vortex;
  • picofuge;
  • magnetic stand;
  • stripes of a paper tape (~5mm wide);
  • forceps
  • Covaris S2
    • change water;
    • start degassing;
    • prepare post-PCR tube holder


[править] Buffers

  • degas for ~15 min Deposition Buffer:
1,2ml + VD per library
  • (optional): TEX- buffer (~0,5 ml per library)
  • (optional): Slide Storage buffer


[править] Libraries

for each library:

  • calculate the volume/ amount of beads for deposition;[3]
  • prepare and label 1.7ml Lo- Bind tubes: #lib — P2-enriched beads


[править] Protocol

  1. vortex P2-enriched beads (20 sec.; be sure, that there is no pellet on the bottom) and pulse-spin;
  2. sonicate two times:
    ○○   Covaris S2: «Covalent Declump 1»/2 (vortex and pulse spin in the middle of program);
    ○○   vortex and pulse-spin;
  3. quantitate beads;
  4. put calculated volume of beads into 1.7ml tube #lib;
  5. magnetic rack (1 min.) and remove supernatant;
  6. wash by Deposition Buffer (three times):
    --- beads tend to stick to the walls, SLOWLY aspirate the supernatant, FROM THE MIDDLE OF THE LIQUID ---
    --- process one tube after another: before you start aspirating liquid from one tube, place the next in the magnetic rack ---

    ○○○   resuspend in 400µl of Deposition buffer, vortex(20 sec.)and pulse spin;
    ○○○   magnetic rack 1 min.; remove supernatant;

  7. resuspend in VD µl of Deposition Buffer: vortex (20 sec) and pulse-spin;
  8. prepare slides:
    --- NB! handle deposition chamber on a thermoisolated surface ---
    • insert a new slide into slide carrier and fix it;
    • insert slide carrier into preheated deposition chamber and STRONGLY tighten the four screws in a crisscross pattern;
    • return assembly at 37°C (on the metal surface of the 37°C incubator);
  9. for each library:
    • sonicate beads four times:
    ○○○○   vortex P2-enriched beads (5 sec) and pulse-spin;
    ○○○○   Covaris S2: «Covalent Declump 3»;
    • pulse-spin;
    • re-suspend by pipetting;
    • deposit beads (corner-by-corner in lower position); pipett only in one way and DO NOT aspirate
    • STRONGLY attach adhesive paper squares;
    • return assembly to 37°C;
  10. incubate at 37°C for at least 1,5 hours;

    • (optional) if >10µl of beads remain after deposition:

    1. calculate the number of beads,
    2. wash with 400µl TEX,
    3. resuspend in 40µl TEX,
    4. store at 4°C


  11. (optional)if sequencing will be done next day: substitute Deposition Buffer on Slide Storage Buffer (leave buffer in the deepening of the deposition chamber). If longer: transfer slide into Storage Chamber with Slide Storage buffer. Store at 4°C




[править] Notes

    • wash deposition chamber
      • wash with bidest. H2O (wipe it with kimwipes clean)
      • shortly wash the chamber in 70% EtOH)
      • finally wash it in bidest. H2O
      • predry with Kimwipes and then put it in the 37°C incubator
  1. should be calibrated for each pair of «top and bottom» of deposition chambers
    • keep preselected pairs of «top and bottom» of deposition chambers;
  2. calculate number of beads to load
    Deposition chamberWFA(million beads)Sequencing (million beads)
    1- well 700
    4- well15 
    8- well  

 


[править] Sequencing slides

  • both surfaces are equivalent;
  • before deposition slides should ALWAYS be DRY. Stored slides in dry exsiccator in a box with holes or not tightly closed. It is a good idea to have large stock in exsiccator in the cold room, and working portion of slides in an exsiccator in the post-PCR room on RT.
  • after deposition slides should ALWAYS be WET. Stored in Storage Chamber or in plastic container filled with Slide Storage Buffer at 4°C. They are stable for weeks or months. Re-sequencing of the same slide is possible, though gives somehow lower quality
  • before removing the slide from the Deposition Chamber, be sure, that Slide Storage Chamber is available or instrument is ready for installation



  • slide characteristics:
WellsNo. of panelsDep. volume[1]QC-run [mln]Sequencing [mln]
Normal dens.High dens.
1~2357-~410µl165283
449515~135µl4x 30 (~3/4)52
8221-~65µl8x 14 (~2/3)24
  1. should be calibrated for each pair of «top and bottom» of deposition chambers


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