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Beads preparation - normal practice

Comments

NB! post-PCR operation.

stage time preparation to ePCR 2h ePCR 3h bead wash 2h bead enrichment 4h 3'-end modification 2h-ON






• libraries:
  • "#lib" in the protocol - name of the library;
  • mean sizes of libraries:
       fragment: ~120-160bp;
       M-P: 156bp;



• tubes:
  • use low-binding tubes (Eppendorf) in the protocol;
  • 1,7ml tubes used in this protocol for Covaris sonication (instead of 0.5ml in the AB-manual);



• beads:
  • it is important, that beads were always wet;
  • pulse-spin is just 1 second;
  • time for magnetic concentration is different for different buffers: 0,5-1 min for 1xTEX; 2 min for Bead deposition buffer, 3 min centrifugation in 60% glycerol;
  • beads are stored in 1xTEX solution at 4°C (after ePCR; P2-enriched; 3'-modified): good quality for more than 6 months;
  • Bead Quantitation:
    • wash NanoDrop with water;
    • use as a reference solution 2µl of the bead diluting buffer;
    • read A₆₀₀ optical density for 2µl of beads. For 1xTEX: A₆₀₀ x 1.1 = ____x10⁶ [beads/µl]