Bead wash

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This protocol is for beads, collected from ONE plate / TWO platesTWO plates / ONE plate

Содержание


NB! post-PCR operation


[править] Equipment and consumables

  • check, that you have:
» waste bottle for butanol-oil phase under the fume hude;
» plastic box with paper towel;
» magnetic stand;
» LoBind tubes;
» bufers:

1 plate
BWB (1X Bead Wash Buffer)2.4ml
TEX (1xTEX bufer)0.5ml
Butanol20ml

2 plates
BWB (1X Bead Wash Buffer)3ml
TEX (1xTEX bufer)1ml
Butanol40ml


  • label Emulsion Collection Tray (ECT): #lib, 1 per plate (per library?)
  • prepare 2x 1.7ml #lib LoBind tubes for each library;


[править] Bead wash

  1. collect emulsion from PCR plates:
    collect beads from both ePCR plates into one TB-cover
    • spin down PCR plates 5 min, 600x g, 20°C;
    • place ECT on the top of PCR plate;
    • prepare everything (balance rotor, insert program, etc.), then flip assembly and centrifuge 1 min, 40x g, 20°C;


  2. remove out-of-emulsion beads:
    • pour emulsion into a 50ml tube;
    • wipe ECT border with a piece of paper towel and close ECT by PCR plate;
    • centrifuge again: 1 min, 40x g, 20°C;
    • transfer the rest of emulsion with 1ml pipette into the 50ml tube;
    • centrifuge 50ml tube 10 min, 600x g, 20°C;
    • fix beads on the bottom of the tube using magnet;
    • pour emulsion only (without beads on the bottom) into a "new 50ml tube";
    • accurately (without mixing of phases) add butanol to a "new 50ml tube" up to 2545ml;
    • centrifuge the "old 50ml tube" again: 5 min, 600x g, 20°C;
    • transfer the rest of emulsion by 1ml pipette to the "new 50ml tube";
    • use ~0.5ml of butanol from the "new 50ml tube" 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) ~200µl 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 and transfer beads into 1.7ml #lib tube;
    • accurately wash the bottom of the 50ml #lib tube with another 600µl of BWB and collect fractions;

  5. BWB washing:
    • prepare a new 1.7ml #lib tube with 1ml of BWB;
    • vortex beads (30 sec);
    • centrifuge 1 min at 14x kg;
    • 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 and transfer to 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 14x kg;

  6. two times TEX washing:
    ○○   magnetic rack (1 min) and remove supernatant;
    ○○   add 200400µl TEX;
    ○○   vortex 20 sec and pulse-spin;

  7. keep at +4°C


Источник — «http://molbiol.ru/wiki/Bead_wash»


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