Daniel:Notebook/ComboLock/2017-3-13

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Revision as of 15:55, 13 March 2017 by >Djacobse (Created page with "=Bolt Test (Started Wednesday March 8)= Back to Calendar ==Protocol== '''Sample Matrix''' {| class="wi...")
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Bolt Test (Started Wednesday March 8)

Back to Calendar

Protocol

Sample Matrix

Sample Condition
Sample 5 (AB) No Ligase
Sample 6 (AB) Normal
  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 Exp uL Total No C uL Total NP uL Total
      PCCA-03 (10 uM) 1 4 0 2
      PCCB-03 (10 uM) 1 4 0 0
      Mirror BC-v2 (10 uM) 1 0 0 2
      Wash buffer 3 12 10 6
      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
    6. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    7. Repeat wash step above
    8. Repeat wash step above