Daniel:Notebook/ComboLock/2016-6-2

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Antibody Hybridization Test[edit]

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Time to test out the antibody binding protocol like I did with C-probes

Protocol[edit]

  1. Mix PLA Buffer
  2. Reagent Stock Conc. Final Conc. Dilution Volume for 1 mL solution (uL)
    BSA* 50 mg/mL (5%) 0.10% 50 20
    Tween 20 100% 0.05% 2000 0.5
    Salmon Sperm DNA 10 mg/mL 0.1 mg/mL 100 10
    EDTA 500 mM 5 mM 100 10
    PBS 10X 1X 10 100
    nfH2O -------- -------- -------- 859.5
  3. Hybridization
    1. Split methanol-frozen cells into 4 (1.25 mL methanol each)
    2. Pellet cells via centrifugation at 600g for 3 min
    3. Add PLA mix and probes to create hybridization solution
    4.   uL PA Mix uL PB Mix uL PLA buffer
      Sample 1 0.5 0 2.5
      Sample 2 0 0.5 2.5
      Sample 3 0.5 0.5 2
      Sample 4 0 0 3
    5. Mix 1 uL cell lysate with 3 uL Probe mix
    6. Incubate 1.5 hours at RT
    7. Add 200 uL 0.05% Tween 20 in PBS
    8. Wash twice with 0.05% Tween 20 in PBS
      1. Pellet cells at 600xg for 3 min
    9. Resuspend samples in 50 uL 1X PBS
  4. qPCR
    1. Set up qPCR reaction according to table
    2. Sample Lane Cell Sample Cells 2X Kapa SYBR qPCR MM 100 uM Primer2 100 uM Primer4-RC 100 uM Primer6 100 uM Primer12-RC H2O Total Volume (uL)
      Sample 1 (PA) A1 S1 1 25 0.5 0.5 0 0 23 50
      Sample 2 (PB) A2 S2 1 25 0 0 0.5 0.5 23 50
      Sample 3 (PA+PB) A3 S3 1 25 0.5 0.5 0.5 0.5 22 50
      Sample 4 (No Probe) A4 S4 1 25 0.5 0.5 0.5 0.5 22 50
      No Template A5 NA 0 25 0.5 0.5 0.5 0.5 23 50
      Mismatch Primers A6 S3 1 25 0.5 0 0.5 0 23 50
      No Primers A7 S3 1 25 0 0 0 0 24 50
      Primers + Antibodies A8 NA 1 25 0.5 0.5 0.5 0.5 22 50
    3. qPCR Cycles
      1. 95C 3 min
      2. 95C 3 sec
      3. 55C 30 sec
      4. 72C 20 sec
      5. plate read
      6. goto b x20
      7. 72C 2 min
      8. 16C hold

    qPCR Results[edit]