Daniel:Notebook/PosSequencing/2017-6-21: Difference between revisions

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[[Daniel:Notebook/PosSequencing|Back to Calendar]]
[[Daniel:Notebook/PosSequencing|Back to Calendar]]
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 [https://tools.thermofisher.com/content/sfs/manuals/MAN0007720.pdfdigital qPCR] to amplify the beads.


==Protocol==
==Protocol==

Revision as of 16:28, 21 June 2017

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 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 (20.2X)
      Primer 2 (10 uM) 4 80.8
      Barcode Oligo 100 pM 5 101
      Beads 4 0
      Kapa 2X SYBR Fast Mix 20 404
      nfH2O 7 141.4
      Total 40 727.2
    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. File:PlateLayout-BeadsqPCR-20170621.png
    6. Vortex and spin down after sealing wells
    7. Use the standard thermocycler program->40 cycles