Bead enrichment

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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:
» 45°-cut 200µl tips;
» magnetic stand;
» LoBind tubes and tips;
» rotator at NT;
» 61°C water bath;


  • prepare Covaris S2:
» change water;
» start degassing;
» prepare post-PCR tube holder;


  • melt, vortex, spin-down and put in ice 1mM Enrichment Oligo (should not be diluted) 3.5µl per plate;


  • prepare buffers (in advance — if more than 4 libraries are processed; otherwise — during EB-charging):
60% Glycerol
fresh weekly: vortex to mix well, store in ice

1 plate2 plates
100% glycerol (1.262g/ml)0.95g / 0.75ml1.9g / 1.5ml
1x TEX buffer0.25ml0.5ml
LS buffer0.25ml0.5ml
total:1.25ml2.5ml


DenBuffer
fresh weekly: mix well, store at NT

1 plate
Denaturing Buffer1.98ml
Denaturant220µl
total:2.2ml

2 plates
Denaturing Buffer3.6ml / 3.62g
Denaturant400µl
total:4ml



  • check, that you have enough buffers:

1 plate
1x TEX buffer9ml
LS buffer0.2ml
Bi-buf2.2ml

2 plates
1x TEX buffer16ml
LS buffer0.4ml
Bi-buf3.6ml


  • prepare 1.7ml tubes, for each library:
» 4x with 300µl 6x with 400µl with 60% Glyc., labelled #lib;
» 26x with 1ml of TEX, labelled #lib




[править] E-beads charging

  1. E-beads aliquoting:
    • vortex E-beads (30 sec);
    • put 650µl1.3ml of E-beads into 1.7ml tube #EB_lib;
    • centrifuge 1 min at 14x kg and remove supernatant;

  2. two times Bi-buf washing:
    ○○   re-suspend E-beads in 900µl1.3ml of Bi-buf: vortex 20 sec;
    ○○   centrifuge 1 min at 14x kg and remove supernatant;

  3. charging by Enrichment Oligo:
    • re-suspend E-beads in 350700µl of Bi-buf: vortex E-beads 20 sec and pulse-spin;
    • add 3.57µl of 1mM Enrichment Oligo;
    • vortex E-beads 30 sec;
    • rotate E-beads at NT for 30 min:
    » start-time: _________
    » finish-time: _________

  4. TEX washing:
    • centrifuge 1 min at 14x kg and remove supernatant;
    • two times TEX washing:
    ○○   re-suspend E-beads in 900µl1.3ml of TEX: vortex 20 sec;
    ○○   centrifuge 1 min at 14x kg and remove supernatant: pour out major part, remove the rest with a pipette;

  5. resuspend E-beads in 150300µl of LS: vortex 30 sec and pulse-spin



  6. [править] Preparation of single-stranded P1 beads

  7. pulse-spin, magnetic rack (1 min) and remove supernatant;
  8. two times denaturation:
    ○○   re-suspend the beads in 300600µl DenBuffer: vortex 20 sec;
    ○○   incubate 1 min and pulse-spin;
    ○○   magnetic rack (1 min) and remove supernatant;

  9. two times washing:
    ○○   re-suspend the beads in 300600µl of TEX: vortex 20 sec and pulse-spin;
    ○○   magnetic rack (1 min) and remove supernatant;

  10. re-suspend the beads in TEX:
    • add 150300µl of TEX;
    • vortex 20 sec and pulse-spin;



    [править] Hybridization

    NB! NO ANY MAGNETS during Hybridization
    NB! it is convenient to process two-tubes portions


  11. declump beads:
    • vortex 20 sec and pulse-spin;
    • Covaris S2: 2x "Covalent Declump 1"/2: vortex and pulse-spin in the middle of the program;
    • vortex and pulse-spin;

  12. combine P1- and E-beads:
    • add E-beads to P1-beads in correspondent #lib tubes;
    • vortex (30 sec) and pulse-spin;
    • Covaris S2: "Covalent Declump 3";
    • shake down (do not spin!);

  13. hybridization of P1- and E-beads:
    • incubate at 61°C for 15 min, vortexing (20 sec) and shaking down every 5 min:
    time 1:    _______     _______     _______     _______     _______     _______
    time 2:    _______     _______     _______     _______     _______     _______
    time 3:    _______     _______     _______     _______     _______     _______
    • cool beads in liquid ice (2 min);

  14. enrichment:
    • vortex (10 sec) and pulse-spin;
    • mix by pipetting and ACCURATELY load 75100µl on top of the 60% glycerol in 1.7ml #lib tubes;
    • centrifuge 3 min at 14x kg;
    • using one "45°-cut" tip per library collect TOP layers from the hinged sides of #lib tubes with glycerol and transfer them to BOTTOMS of twosix #lib tubes with 1 ml of TEX;
    • rinze the tip by pipetting upper part of TEX buffer;

  15. TEX washing:
    • vortex #lib tubes (20 sec);
    • centrifuge 1 min at 14x kg and remove supernatant;
    • resuspend the beads in 400300µl of TEX: vortex 20 sec;
    • collect in two 1.7ml tubes;
    • centrifuge 1 min at 14x kg and remove supernatant;


  16. re-suspend the beads in DenBuffer:
    • resuspend by pipetting the beads in 100µl of DenBuffer;
    • combine contents of two #lib tubes in one;
    • use additional 100µl DenBuffer to wash tubes and tip
    NB! MAGNETIC RACK starts here




    [править] Washing

  17. denaturation:
    --- repeat until supernatant is absolutely clean (normally three times — only tiny white blur should be in solution during the second washing, third washing should be practically clean) ---

    ○○○   pulse-spin and magnetic rack (1 min);
    ○○○   ACCURATELY remove supernatant with E-beads;
    ○○○   add 400800µl of DenBuffer and vortex 20 sec;
    ○○○   incubate 1 min;

  18. two times TEX-washing:
    ○○   re-suspend the beads in 400800µl of TEX: vortex 20 sec and pulse-spin;
    ○○   magnetic rack 1 min and remove supernatant;

  19. declumping:
    • resuspend the beads in 400800µl of TEX: vortex 1 min and pulse-spin;
    • Covaris S2: "Covalent Declump 1";
    • pulse-spin;

  20. secondary TEX-washing:
    --- repeat until supernatant is absolutely clean (at least two times) ---

    ○○   magnetic rack 1 min and remove supernatant;
    ○○   re-suspend the beads in 400800µl of TEX: vortex 20 sec and pulse-spin;




[править] Notes

  • beads should have ~95% purity after enrichment;


  • charged Enrichment Beads should be used within one week (store in TEX at 4°C);


  • removal of supernatant from uncharged Enrichment beads should be done in three steps:
» centrifuge 1 min at 14x kg;
» turn tubes in the rotor on 180° to put pellet down;
» wait 1.5 min until beads went down;
» remove ~80% of supernatant (until beads come to the tip);
» slightly shake tube to wash beads from the wall;
» repeat above procedure twice with shorter centrifugation: 0.5 min at 14x kg;


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