Daniel:Notebook/ComboLock/2017-2-9

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Beads Binding Test[edit]

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Since this should only take a day (now that I have the proper materials), I want to test something: the beads binding. I want to see at what temp the beads lose their binding ability and at what temp we lose C probes for that matter. To that extent, I have planned the following experiment. It will cover incubation temperatures ranging from RT to 90C and will include a No C probe and no Template negative controls. I will also use positive controls in the qPCR (straight C probes and straight template).

Sample Matrix and Workflow[edit]

Sample Matrix

Sample 1 (AB) 2 (AB) 3 (AB) 4 (AB) 5 (AB) 6 (AB) 7 (AB) 8 (AB) 9 (AB) 10 (AB)
Condition RT No Template 30C 40C 50C 60C 70C 80C 90C No C Probe


Workflow Diagram

File:20170208-BeadsTest-Workflow.png

Protocol[edit]

  1. Template-Bead Binding
    1. Suspend 2 uL (8 ug) beads per sample in 100 uL wash buffer
    2. Apply magnet for 30 sec and remove supernatant
    3. Add 2 uL 10 uM template oligo per sample to bead solution; incubate at RT for 5 min; For "No Template" add 2 uL 2 mg/mL BSA-biotin
    4. Wash beads with 100 uL wash buffer; vortex to suspend; apply magnet and remove supernatant
  2. C Probe Hybridization
    1. Combine following into a 0.2 mL tube
    2. Reagent uL per Sample uL Total (Samples+NT) uL Total (No C Probe)
      PCCA-03 (10 uM) 1 18 0
      PCCB-03 (10 uM) 1 18 0
      Wash buffer 3 54 10
      Total 5 90 10
    3. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    4. Add 5 uL probe mixture to beads
    5. Incubate at 40C for 1 hour with agitation
  3. Wash Testing
    1. Repeat following washes 5 times
      1. Add 100 uL wash buffer
      2. Set on magnet for 1 min; take 15 uL aside for testing
      3. Vortex and incubate for 5 minutes at appropriate incubation temperature
      4. Set on magnet for 1 min; remove supernatant completely

Ran out of time; continued tomorrow