Daniel:Notebook/ComboLock/2017-2-9

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Revision as of 17:37, 9 February 2017 by >Djacobse (Created page with "=Beads Binding Test= Daniel:Notebook/ComboLock/Back to Calendar Since this should only take a day (now that I have the proper materials), I want to test something: the b...")
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Beads Binding Test

Daniel:Notebook/ComboLock/Back to Calendar

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

Sample Matrix


Workflow Diagram

File:20170208-BeadsTest-Workflow.png

Protocol

  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
    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 Phosphate) uL Total (No C Probe)
      PCCA-03 (10 uM) 1 4 2 0
      PCCB-03 (10 uM) 1 4 2*** 0
      Wash buffer 3 12 6 10
      Total 5 20 10 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