Bead deposition, SOLiD 4

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NB! post-PCR operation

Содержание

[править] Prepare in advance

Foto of deposition chamber
* notched side is the bottom of the slide

[править] Equipment and consumables

Kits required

  • SOLiD TM XD Slide Kit — 4x2 Sequencing Slides, # 4445812 — slides (stored at −20°C)
  • SOLiD TM XD Slide Kit — Slide Storage Buffer, #4451927 — storage buffer
  • SOLiD TM XD Slide Kit — Deposition Buffer, #4448388 — deposition buffer
  • SOLiD TM XD Slide Kit — Prep Reagent, #4448390

slides:

  • Remove XD slides from −20°C storage to equilibrate to RT;

check that you have:

  • vortex;
  • picofuge;
  • magnetic stand;
  • stripes of a paper tape (~5mm wide);
  • pincette
  • kimwipes or other dust-free paper towels

prepare Covaris S2 in the post-PCR room:

  • change water;
  • start degassing;
  • prepare post-PCR tube holder


[править] Deposition chambers

  • NB! for SOLiD 3+ use Solid Opti Slide Carrier (marked with «OPTI» on the top and with a blue side)
  • Clean (link to the washing protocol in the neighbour protocol) the seleczed DC and slide carriers;
  • dry out at 37°C for several hours or ON (put on the metal surface of the 37°C incubator)
  • DC should be RT for deposition


[править] Buffers

  • degas Deposition Buffer for ~15 min:
1.2 ml + VD per library
  • (optional): TEX buffer (~0.5ml per library)
  • (optional): Slide Storage buffer


[править] Libraries

for each library:

  • write on the card deposition volumes VD [1] of preheated chamber;
  • calculate the volume of beads for deposition;
  • prepare and label 1.7ml eppendorf 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 the program);
    ○ ○ vortex and pulse-spin;
  3. quantitate beads (link to the protocol);
  4. put calculated volume of beads into 1.7ml tube #lib;
  5. magnetic rack (1 min) and remove supernatant;
  6. wash by XD Slide Deposition Buffer v2 (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 strart aspirating liquid from one tube, place the next is in the magnetic rack ---
    ○ ○ ○ resuspend in 400µl of XD Slide Deposition Buffer v2: vortex (20 sec) and pulse-spin;
    ○ ○ ○ magnetic rack 1 min and remove supernatant;
  7. resuspend in VD µl of XD Slide Deposition Buffer v2: vortex (20 sec) and pulse-spin; put in ice
  8. for each library (without pauses):
    prepare slides:
    --- NB! handle deposition chamber on a thermoisolated surface (e.g. block of paper towels)---
    • insert a new slide into SOLiD4 or Opti slide carrier and fix it;
    • put the slide carrier with a flat side down on a clean surface
    • pipette 3ml of SOLiD XD Slide Prep Reagent over the slide to cover it completely, let stand for 2 min
    • NB! Do not let the slide dry out!
    • Slowly decant the reagent off the surface onto kimwipes or paper towel
    • Wash the slides with XD Slide Deposition Buffer v2:
    ○ ○ ○ pipette 3ml of XD Slide Deposition Buffer v2 over the slide to cover it completely, let stand for 2 min;
    ○ ○ ○ decant the reagent off the surface onto kimwipes or paper towel;
    • Immediately insert slide carrier into corresponding deposition chamber , STRONGLY tighten the four screws tabs in a crisscross pattern
    • sonicate beads four times:
    ○ ○ ○ ○ vortex P2-enriched beads (5 sec) and pulse-spin;
    ○ ○ ○ ○ Covaris S2: «Covalent Declump 3»
    • pulse-spin shortly
    • with 1ml pipette resuspend beads by pipetting
    • deposit beads (corner-by-corner in lower position);
    • remove excess of the liquid from loading holes by 10µl tip;
    • STRONGLY attach adhesive paper squares;
    • centrifuge the slides in Deposition Chambers at 167x g for 10 min
    • incubate at RT for about 1hour
  9. (optional) if >10µl of beads remain after deposition: i. calculate the number of beads
    ii. wash with 400µl TEX,
    iii. resuspend in 40µl TEX,
    iv. store at 4°C
  10. (optional) if sequencing will be done next day: substitute Deposition Buffer on Slide Storage Buffer (leave buffer in the deepening of the deposition chamber) and put at 4°C. For longer storage: transfer slide into Storage Chamber with Slide Storage buffer. Store at 4°C


[править] Notes


[править] Sequencing slides

  • both surfaces are equivalent;
  • before deposition slides should ALWAYS be DRY.
  • during surface modification and deposition slides should ALWAYS be WET.
  • before removing the slide from the Deposition Chanber, be sure, that Slide Storage Chamber is available or instrument is ready for installation


[править] References

  1. should be calibrated for each pair of «top and bottom» of deposition chambers


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