Daniel:Notebook/ComboLock/2017-6-14

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Antibody Detection Test-Positive Control[edit]

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This will be a basic test of the antibody detection.

Protocol[edit]

Sample Matrix

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No Antibodies
  1. Template-Bead Binding
    1. Suspend 2.5 uL (25 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Prepare following mixes
      1. 4X mix (samples 1,3): 80 uL Dynabuffer, 72 uL nfH2O, 8 uL BSA-biotin 0.2 mg/mL (6 pmol, 6X excess)
      2. 2X mix (sample 2): 40 uL Dynabuffer, 36 uL nfH2O, 4 uL BSA-biotin 0.2 mg/mL (6 pmol, 6X excess)
    4. Add 40 uL per sample and incubate at RT for 15 min
    5. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
    ******
    
  2. Antibody hybridization
    1. Combine following into 0.2 mL tubes
    2. Reagent uL per Sample Exp uL Total (4X 1,2) No Ab uL Total (2X-Sample 3)
      Ab-A (75 ug/mL-0.5 uM) 2 8 0
      Ab-B (75 ug/mL-0.5 uM) 2 8 0
      PBS+EDTA (2X+10 mM) 10 40 20
      nfH2O 6 24 20
      Total 20 80 20
    3. Add probes to cell sample for final concentration of 0.5 nM
    4. Incubate 1.5 hours at RT
    5. Wash beads with 100 uL wash buffer with 0.05% Tween 20; apply magnet and remove supernatant
    6. Repeat wash step above
    7. Repeat wash step above

Protocol-Part 2[edit]

  1. Latch and Bolt Hybridization/Ligation
    1. Set up the following master mix
    2. Reagent Stock Conc Final Conc./Amt uL added Master Mix (6.2X)
      T4 Ligase Reaction Buffer 5X 1X 4 24.8
      Latch Oligo 10 uM 10 pmol 1 6.2
      Bolt Oligo 10 uM 10 pmol 1 6.2
      Dephosphorylated Bolt 2.5 uM 10 pmol 8 0
      nfH2O NA NA 13 80.6
      Total     19 117.8
    3. Add 19 uL probe mix to each sample
    4. Incubate at 55C for 10 minutes
    5. Incubate at 25C for 10 minutes
    6. Lower the temperature to 20C; wait 30 seconds then take off incubator
    7. Add 1 uL T4 Ligase to each reaction
    8. Incubate at 16C overnight
    9. Continued tomorrow