Daniel:Notebook/PosSequencing/2017-6-22

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Beadless emPCR[edit]

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This is to test the emPCR without using beads.

Protocol[edit]

  1. Oil phase
    1. Mix 5 uL Triton X, 40 uL Tween-80, and 450 uL Span 80 into 2 mL Mineral oil and 8 mL Tegosoft DEC
    2. Vortex vigorously to mix
  2. Aqueous Phase Preparation
    1. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    2. Repeat wash step
    3. Resuspend beads in 10 uL TE buffer
    4. Mix ingredients using the following table
    5. Component uL Added uL Added Final Concentration
      DreamTaq 10X PCR Buffer (20 mM MgCl2) 10 10 1X
      10 mM dNTPs 10 10 1 mM
      25 mM MgCl2 6 6 3.5 mM
      10 uM Primer A (Free primer-Primer 2) 10 10 1 uM
      10 uM dual biotin primer 2 2 0.2 uM *
      Hydrogel beads, 2M/uL 0 0 0
      DreamTaq Polymerase (5 U/uL) * 2 2 10 Units
      Barcode Oligo v1 (template, 100 pM, 100 nM) 5 5* 10 pM, 10 nM*
      100 mM Ammonium Sulfate 5 5 5 mM
      1 mM Spermidine*** 10 10 100 uM
      TIPP 0 0 0
      Water 45 45 NA
      Total Volume 100 100  
      • Note: This is based off normally adding in 10 uL of beads containing 12.5 pmol biotinylated primer + 0.5 uL 10 uM primer = 17.5 pmol =0.175uM final concentration. The increased aqueous addition makes the final concentration 0.2uM
  3. Oil-Aqueous Mixing
    1. Mix 10 uL aqueous into 100 uL oil mixture
    2. Vortex for 30 seconds
    3. Repeat mixing to 100 uL (5 times)
    4. Mix with vortexer for 3 minutes
    5. Dilute with pure mineral oil for imaging (1:100)
    6. Distribute 150 uL total volume into 3 tubes, 50 uL each; use same pipette tip
  4. Emulsion PCR
    1. Perform PCR with the following settings
      1. 95C 2 min
      2. 95C 20 sec
      3. 45C 30 sec
      4. 70C 30 sec
      5. Goto b 50 times
      6. 4C forever

Continued tomorrow