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preparation to ePCR2h
bead wash2h
bead enrichment4h
3'-end modification2h-ON

[править] Libraries

  • "#lib" in the protocol - name of the library
  • mean sizes of libraries:
M-P (Eco15I)156bp

[править] 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)

[править] Beads

  • it is important, that beads were always wet
  • pulse-spin is just 1 second
  • time for magnetic concentration is different for different buffers:
1xTEX0.5-1 min
Bead deposition buffer2 min
60% glycerol3 min
  • beads are stored in 1xTEX solution at 4°C (after ePCR; P2-enriched; 3'-modified): good quality for more than 6 months

  • Bead Quantitation
» declump beads: "Covalent Declump 3"
» wash NanoDrop with water
» select "cell culture" measurement program A600
» use as a reference solution 2µl of the bead diluting buffer
» read A600 optical density for 2µl of bead suspension
if 1xTEX is used as a dilution buffer, beads concentration is:
concentration  =  A600 * 1.1 * 106 [beads/µl]
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