Matt:LabNotes/2015-7-1: Difference between revisions
Jump to navigation
Jump to search
>Mzcai |
>Mzcai mNo edit summary |
||
Line 10: | Line 10: | ||
===DARTFISH with suppv2=== | ===DARTFISH with suppv2=== | ||
====Sample labeled "DARTFISH 7.1.2015"==== | |||
#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''' | ||
Line 31: | Line 30: | ||
#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 | ||
#Store at 4C<!-- | #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) | |||
====Continued protocol with 2 dishes==== | |||
<!-- | |||
#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 00:55, 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)