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 |
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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 - | #*DEPC-H2O - 159ul | ||
#*M-MuLV RT Buffer 10X - 20ul | #*M-MuLV RT Buffer 10X - 20ul | ||
#*dNTP 25mM - 2ul | #*dNTP 25mM - 2ul | ||
#*aa-dUTP | #*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)
- Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015
- Follow protocol from last time
- 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
- Use 1000X concentration of suppressor oligos to match in vitro experiment
DARTFISH with suppv2
- Sample labeled "DARTFISH 7.1.2015"
- Wash the cells with 1ml of PBS (RNase free) 3 times
- 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
- Add RT mixture and incubate for 10min at 4C
- Transfer sample to 37C overnight (~18hrs)