Daniel:Notebook/PosSequencing/2017-6-21

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Revision as of 17:12, 22 June 2017 by >Djacobse (→‎Discussion)
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Bead qPCR Test

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This experiment has 2 purposes: to determine the density of beads necessary to optically block qPCR detection (and hopefully some levels that don't) as well as the efficiency of bead-based qPCR vs aqueous. This will hopefully help me make the next experiment run smoother, specifically using digital qPCR to amplify the beads.

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) ~Amt BioU Primer (pmol)
      10 M 0 2 5 pmol
      2 M 1 4 1 pmol
      1 M 1 2 500 nmol
      0.2 M 2 4 100 nmol
      0.1 M 2 2 50 nmol
      20 K 3 4 10 nmol
      10 K 3 2 5 nmol
      1 K 4 2 1 nmol


  3. qPCR
    1. Make the following master mix
    2. Reagent 1X Added Master Mix (21.2X)
      Primer 2 (10 uM) 1 21.2
      Barcode Oligo 100 pM 1 21.2 (1)
      Beads 4 84.8
      Kapa 2X SYBR Fast Mix 20 424
      nfH2O 14 296.8 (155.4)
      Total 40 848
    3. Add 36 uL master mix to each lane
    4. Add n uL of sample and 4-n uL nfH2O to appropriate lanes according to plate layout
    5. For the "Supernatant" sample add 10 uL TE to original beads (0 dilution), mix to suspend, then pull down; take 4 uL supernatant for sample
    6. File:PlateLayout-BeadsqPCR-20170621.png
    7. Vortex and spin down after sealing wells
    8. Use the standard thermocycler program->40 cycles

Results

Discussion

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