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Bead deposition
Equipment and consumables
notched side is the bottom of the slide
- in the previous day prepare deposition chambers:
- clean;
- preheat at 37°C for several hours (on the metal floor of the 37°C incubator);
- Covaris S2:
- change water;
- start degassing;
- prepare post-PCR tube holder;
- for each library:
- write on the card deposition volumes VD of preheated chamber;
- calculate the volume of beads for deposition;
- prepare and label 1,7ml eppendorf tubes: #lib - P2-enriched beads;
- degas for ~15 min Deposition Buffer (0.6ml per library for QC, {0.5ml + VD} for sequencing);
- prepare a stripe of a paper tape (~5mm wide);
- check, that the large centrifuge is not cold, set: 37°C, 150x g, 5 min, put plate-holders on the metal floor of the 37°C incubator;
- equipment:
- vortex;
- picofuge;
- magnetic stand;
- screwdrivers for deposition chamber;
Protocol
- vortex P2-enriched beads (20 sec, be sure, that there is no pellet on the bottom) & pulse-spin;
- sonicate two times:
◊ ◊ Covaris S2: "Enrich-01"/2;
◊ ◊ vortex and pulse-spin;
- for NON- quantitated beads: quantitate them;
- put calculated volume of beads into 1.7ml tube #lib;
- magnetic rack (1 min) & remove supernatant;
- wash by Deposition Buffer (three times):
--- beads tend to stick to the walls, SLOWLY aspirate the supernatant, NOT FROM THE SURFACE ---
--- process one tube after another: when aspirate liquid from one tube, the next is in the magnetic rack ---
◊ ◊ ◊ re-suspend in 150µl of Deposition Buffer: vortex (20 sec) & pulse-spin;
◊ ◊ ◊ magnetic rack 1 min & remove supernatant;
- re-suspend in VD µl of Deposition Buffer: vortex (20 sec) & pulse-spin;
- prepare slides:
--- NB! handle deposition chamber on a thermoisolated surface ---
- insert a new slide into slide carrier & SLIGHTLY fix it;
- insert slide carrier into preheated deposition chamber & STRONGLY DIAGONALLY tighten four screws;
- return assembly at 37°C (on the metal floor of the 37°C incubator);
- for each library:
- sonicate beads four times:
◊ ◊ ◊ ◊ vortex P2-enriched beads (5 sec) & pulse-spin;
◊ ◊ ◊ ◊ Covaris S2: "Enrich-01"/2;
- pulse-spin;
- re-suspend 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;
- return assembly to 37°C;
- (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;
- centrifuge assembly at 150xg for 5 min (assembly should be on a thermoisolated surface);
- incubate deposition chamber at 37°C for at least 2.5-3 hours;
- if sequencing will be done next day: substitute Deposition Buffer on Slide Storage Buffer (some buffer in deposition wells). If longer: transfer slide into Storage Chamber with Slide Storage buffer. Store at 4°C
Notes
- tubes: see Notes in "ePCR";
- beads: see "ePCR";
- sequencing slides:
- both surfaces are equivalent;
- before deposition slides should be ALWAYS DRY. Stored in dry: open box, take the rack, open rack, take slide, DO NOT CLOSE the rack, put it back opened. It is a good idea to put a portion of slides in separate box. They are good for several months after opening;
- after deposition slides should be ALWAYS WET. Stored in Storage Chamber or in plastic container filled with Slide Storage Buffer at 4°C. They are stable for weeks or months. Second re-sequencing of the same slide gives somehow lower quality;
- before removing the slide be sure, that Slide Storage Chamber is available or instrument is ready for installation;
- deposition chambers:
- washing: only mQ water or 70% ethanol; NO DETERGENTS;
- keep preselected pairs of "top & bottom" of deposition chambers;
- slide characteristics:
| Wells | No. of panels | Dep. volume* | QC-run [mln] | Sequencing [mln] |
| Normal dens. | High dens. |
| 1 | ~2357 | - | 410µl | 165 | 283 |
| 4 | 495 | 15 | ~135µl | 4x 30 (~3/4) | 52 |
| 8 | 221 | - | ~65µl | 8x 14 (~2/3) | 24 |
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* should be calibrated for each pair of "top & bottom" of deposition chambers
Second-generation sequencing
URL: http://seq.zbio.net
e-mail: soldatov@molgen.mpg.de
visits:
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Second-generation sequencing > Bead deposition
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