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)
- 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)
- 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
- Center of glass was dry and there was solution around edges of dish
- 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
Sample labeled "DARTFISH2 7.2.2015"
- Because first sample had dry areas after RT preparing a second sample just in case
- 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)
- 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