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

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>Mzcai
(Created page with "==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 **...")
 
>Mzcai
Line 14: Line 14:
#Wash the cells with 1ml of PBS (RNase free) 3 times
#Wash the cells with 1ml of PBS (RNase free) 3 times
#Prepare RT mixture '''on ice'''
#Prepare RT mixture '''on ice'''
#*DEPC-H2O - 157ul
#*DEPC-H2O - 159ul
#*M-MuLV RT Buffer 10X - 20ul
#*M-MuLV RT Buffer 10X - 20ul
#*dNTP 25mM - 2ul
#*dNTP 25mM - 2ul
#*aa-dUTP 2mM - 4ul
#*aa-dUTP 4mM - 2ul
#*RT primer 100uM - 5ul
#*RT primer 100uM - 5ul
#**Nonamer
#**Nonamer

Revision as of 22:06, 1 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)