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P1 bead Wash

Equipment and consumables Protocol Notes  Comments

NB! post-PCR operation. This protocol is for beads, collected from ONE plate / TWO platesTWO plates / ONE plate

Equipment and consumables

• vortex;
• TB-covers (covers for 20µl Biozym tip-boxes):
  • label TB-covers according to the libraries #lib;
  • insert Saran-wrap in it & stick it to the walls;
• waste bottle for butanol-oil phase under the fume hude;
• plastic box with paper towel;
• magnetic stand;
• powder-free gloves;
• LoBind tubes & tips;
• prepare 2x 1,7ml #lib LoBind tubes for each library;

1 plate 2 plates 4 plates BWB (1X Bead Wash Buffer) 7ml 15ml 30ml Bi-buf (1X Bind & Wash Buffer) 4ml 8ml 16ml TEX (1xTEX bufer) 8ml 16ml 32ml

Protocol

1. collect beads
   collect beads from both ePCR plates into one TB-cover
   • spin PCR plates 5 min, 600x g, 20°C;
   • place TB-cover on the top of PCR plate;
   • prepare everything (balance rotor, insert program, etc.), then flip assembly & centrifuge 1 min, 50x g, 20°C;
2. remove out-of-emulsion beads:
   • unclip Saran-wrap and transfer emulsion into 50ml tube;
   • spin down emulsion from Saran-wrap: ~3 runs 1 min, 600x g, 20°C;
   • centrifuge 10 min, 600x g, 20°C;
   • collect beads on the bottom of the tube using magnet;
   • pour emulsion only (without water phase) into a "new 50ml tube";
   • add butanol to a "new 50ml tube" up to 2545ml;
   • centrifuge "old 50ml tube" again: 5 min, 600x g, 20°C;
   • transfer the rest of emulsion by 1ml pipette;
   • use butanol to transfer the very rest of emulsion (do not disturb bead pellet);
3. break emulsion:
   • vortex 50ml #lib tube (2 min);
   • centrifuge 5 min 2,000g 20°C;
   • (optional) few ml of aqueous phase (lowest) may be taken for the control of PCR reaction;
   • gently decant butanol-oil-water phase into waste bottle (pellet is quite rigid);
   • put 50ml #lib tube upside down on the fresh paper towel for 5 min;
4. transfer beads into small tube:
   • wipe edge and inside surface with paper towel;
   • accurately add to the bottom 600µl of BWB;
   • soak pellet for 2 min;
   • accurately re-suspend beads by pipetting & transfer beads into 1.7ml #lib tube;
   • accurately wash bottom of 50ml #lib tube with another 600µl of BWB & collect fractions;
5. BWB washing:
   • prepare a new 1.7ml #lib tube with 1ml of BWB;
   • vortex beads (30 sec);
   • centrifuge 1 min at 14,000xg;
   • water pump: aspirate ~3/4 of the supernatant, remove all drops, foam and oil from tube walls;
   • magnetic rack (1 min);
   • remove the rest of supernatant;
   • accurately re-suspend pellet by pipetting in 150300µl of BWB & transfer on the bottom of prepared tube with 1ml of BWB;
   • take ~150µl of clean upper phase and wash tip and the bottom of the previous tube;
   • vortex 20 sec;
   • centrifuge 1 min at 14,000xg;
6. two times TEX washing:
     ◊  ◊   magnetic rack (1 min) & remove supernatant;
     ◊  ◊   add 200400µl TEX;
     ◊  ◊   vortex 20 sec & pulse-spin;
7. keep at +4°C;

Notes

• covers for 20µl tip-boxes: catalog No???;