Matt:LabNotes/2015-7-20

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Revision as of 21:26, 20 July 2015 by >Mzcai (Created page with "==Motor Neurons DARTFISH CA12kNov14_suppv2== *Picked up third batch iPS derived motor neurons from Yeo Lab (Sebastian) fixed on 7/17/2015 at 5pm **Fixed in 4% paraformaldehyde...")
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Motor Neurons DARTFISH CA12kNov14_suppv2

  • Picked up third batch iPS derived motor neurons from Yeo Lab (Sebastian) fixed on 7/17/2015 at 5pm
    • Fixed in 4% paraformaldehyde RT for 15min
    • Permeabilized/store in 70% EtOH at 4C (~72 hours before this DARTFISH experiment)
  • Use 1000X concentration of suppressor oligos to match in vitro experiment
      • e.g. 100nM CA12kNov2014_V4 = 28.5pM per padlock probe, therefore 28.5nM of each suppressor oligo


DARTFISH with suppv2

  • Sample labeled "DARTFISH 7.20.2015"
  1. Wash the cells with 1ml of PBS (RNase free) 3 times
  2. Prepare RT mixture on ice
    • DEPC-H2O - 157ul
    • M-MuLV RT Buffer 10X - 20ul
    • dNTP 25mM - 2ul
    • aa-dUTP 2mM - 4ul
    • RT primer 100uM - 5ul
      • Nonamer
    • RNase inhibitor 40U/ul - 2ul
    • M-MuLV reverse transcriptase 100U/ul - 10ul
  3. Add RT mixture and incubate for 10min at 4C
  4. Transfer sample to 37C overnight (~18hrs)

Imaging