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

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#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
#Add 200ul 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
#Add 200ul 1M Tris (pH 8.0) for 30min at RT
#Wash with 1X PBS twice
#Store at 4C


====Continued protocol with 2 dishes====
====Continued protocol with 2 dishes====

Revision as of 17:02, 3 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)
    • Center of glass was dry and there was solution around edges of dish
      • The vortexer was shaking all night in the incubator and the vibration may disturb the solution
  5. Wash with 1X PBS once
  6. Add 200ul 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
  10. Store at 4C

Sample labeled "DARTFISH2 7.2.2015"

  • Because first sample had dry areas after RT preparing a second sample just in case
  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 200ul 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
  10. Store at 4C

Continued protocol with 2 dishes