Daniel:Notebook/ComboLock/2017-3-1

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Padlock0601 Test

Back to Calendar

Testing the new arrival of padlock0601, which also contains a restriction enzyme cutting site (Bam)

Protocol-Part 1

  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
    6. Wash beads with 100 uL wash buffer; apply magnet and remove supernatant
    7. Repeat wash step above
    8. Add 100 uL cold Low Salt Buffer; apply magnet and remove supernatant

Protocol-Part 2