Daniel:Notebook/PosSequencing/2017-6-21

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Revision as of 15:56, 21 June 2017 by >Djacobse (→‎Protocol)
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Bead qPCR Test

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Protocol

  1. Template-Bead Binding; Make 4 samples and follow this protocol for each
    1. Suspend 5 uL (50 ug, ~50M beads) beads in 100 uL Dynabuffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Suspend in 20 uL Dynabuffer (conc. 2.5 ug/uL)
    4. Add 5 uL 10 uM (50 pmol total, 2X excess) dual biotin oligo and 15 uL nfH2O per sample to bead solution; incubate at RT for 15 min
    5. Wash twice with 100 uL Dynabuffer
    6. Resuspend beads in 10 uL TE buffer
  2. Dilutions
    1. Make dilutions using 2 uL suspended beads with 18 uL TE for dilutions
    2. Make dilutions of 1:10, 1:100, 1:1000, and 1:10000
    3. Sample Matrix
      Beads in Sample Dilution (Power 10) Amount Added (uL)
      10 M 0 2
      2 M 1 4
      1 M 1 2
      0.2 M 2 4
      0.1 M 2 2
      20 K 3 4
      10 K 3 2
      1 K 4 2


  3. qPCR
    1. Make the following master mix