Matt:LabNotes/2015-7-1: Difference between revisions

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#*M-MuLV reverse transcriptase 100U/ul - 10ul
#*M-MuLV reverse transcriptase 100U/ul - 10ul
#Add RT mixture and incubate for 10min at 4C
#Add RT mixture and incubate for 10min at 4C
#Transfer sample to 37C overnight (~18hrs)<!--
#Transfer sample to 37C overnight (~18hrs)
#Wash with 1X PBS once
#Wash with 1X PBS once
#Add 300ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
#Add 300ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
#Wash with 1X PBS twice
#Wash with 1X PBS twice
#Add 200ul 1M Tris (pH 8.0) for 30min at RT
#Add 200ul 1M Tris (pH 8.0) for 30min at RT
#Wash with 1X PBS twice
#Wash with 1X PBS twice<!--
#Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
#Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
#Wash with 2ml H2O twice
#Wash with 2ml H2O twice

Revision as of 17:49, 2 July 2015

Motor Neurons DARTFISH CA12kNov14_suppv2

  • Received Bipolar Neurons (iNGN) from Alex at Harvard
    • Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015
      • RT for 15min
    • Permeabilized/store in 70% EtOH at 4C (~24 hours before this DARTFISH experiment)

DARTFISH with suppv2

  • Sample labeled "DARTFISH 7.1.2015"
  1. Wash the cells with 1ml of PBS (RNase free) 3 times
  2. Prepare RT mixture on ice
    • DEPC-H2O - 159ul
    • M-MuLV RT Buffer 10X - 20ul
    • dNTP 25mM - 2ul
    • aa-dUTP 4mM - 2ul
    • 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)
  5. Wash with 1X PBS once
  6. Add 300ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  7. Wash with 1X PBS twice
  8. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  9. Wash with 1X PBS twice