Daniel:Notebook/ComboLock/2016-12-13

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Revision as of 21:32, 13 December 2016 by >Djacobse (Created page with "=Mirror Probe Test= Back to Calendar This is a continuation of the failed experiment from Friday with a...")
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Mirror Probe Test

Back to Calendar

This is a continuation of the failed experiment from Friday with a corrected version of the template.

Sample Matrix

Sample Condition
Sample 1 (AB) Normal
Sample 2 (AB) No Template
Sample 3 (AB) No C probe

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 1 uL of each C probe (10 uM stock) and 3 uL bead wash buffer into a 0.2 mL tube
    2. Heat C probes to 90C for 5 min; Chill probes on ice to quench
    3. Add 5 uL probe mixture to beads
    4. Incubate at 40C for 1 hour with agitation; start part 3 after incubation begins


    Buffers

    Wash Buffer

    Reagent Stock Final Dilution Amt in 20 mL
    NaCl 1.5 M 0.5 M 3 6.66 mL
    Tris-HCl 500 mM 20 mM 25 800 uL
    EDTA 0.5M 1 mM 500 40 uL
    nf H2O NA NA NA 12.5 mL

    Low Salt Buffer

    Reagent Stock Final Dilution Amt in 10 mL
    NaCl 1.5 M 0.15 M 10 1 mL
    Tris-HCl 500 mM 20 mM 25 400 uL
    EDTA 0.5M 1 mM 500 20 uL
    nf H2O NA NA NA 8.58 mL