Matt:LabNotes/2015-6-19: Difference between revisions
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>Mzcai (Created page with "===DARTFISH with suppv2=== *Sample: 37C 10min **Labeled "DARTFISH 6.19.2015" #Wash the cells with 1ml of PBS (RNase free) 3 times #Prepare RT mixture '''on ice''' #*DEPC-H2O ...") |
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==Motor Neurons DARTFISH CA12kNov14_suppv2== | |||
*[[Matt:LabNotes/2015-6-18 | Picked up second batch iPS derived motor neurons from Yeo Lab (Sebastian)]] | |||
**Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015 | |||
***RT for 15min | |||
**Permeabilized/store in 70% EtOH at 4C (~72 hours before this DARTFISH experiment) | |||
*[[Matt:LabNotes/2015-6-8#DARTFISH | Last time forgot to add suppressor oligos]] | |||
**Use 1000X concentration of suppressor oligos to match [[Matt:LabNotes/2015-5-18 | in vitro experiment]] | |||
***e.g. 100nM CA12kNov2014_V4 = 28.5pM per padlock probe, therefore 28.5nM of each suppressor oligo | |||
===DARTFISH with suppv2=== | ===DARTFISH with suppv2=== | ||
*Sample | *Sample labeled "DARTFISH 6.19.2015" | ||
#Wash the cells with 1ml of PBS (RNase free) 3 times | #Wash the cells with 1ml of PBS (RNase free) 3 times | ||
Line 14: | Line 23: | ||
#*M-MuLV reverse transcriptase 100U/ul - 10ul | #*M-MuLV reverse transcriptase 100U/ul - 10ul | ||
#Add RT mixture and incubate for 10min at 4C | #Add RT mixture and incubate for 10min at 4C | ||
#Transfer sample to 37C overnight (~ | #Transfer sample to 37C overnight (~18hrs) | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 300ul cold BS(PEG)9 ( | #Add 300ul 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 | #Wash with 1X PBS twice | ||
#Add 200ul 1M Tris (pH 8.0) for 30min at RT | #Add 200ul 1M Tris (pH 8.0) for 30min at RT | ||
Line 22: | Line 31: | ||
#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 | ||
#Prepare Ampligase mixture | #Prepare Ampligase mixture '''on ice''' | ||
#* | #*Suppressor Oligos 50nM (labeled supp V4 ALL 5.18.15) - 55ul | ||
#*Ampligase Buffer 10X - | #*Ampligase Buffer 10X - 10ul | ||
#*[[Matt:LabNotes/2015- | #*[[Matt:LabNotes/2015-6-3#Qubit_ssDNA_Quantification | Padlock Probes 438nM]] - 25ul | ||
#**Heat to 90C first and snap cool on ice block | #**Heat to 90C first and snap cool on ice block | ||
#*Ampligase 5U/ul - | #*Ampligase 5U/ul - 10ul | ||
#Add Ampligase mix to sample and incubate 3hr at 60C then | #Add Ampligase mix to sample and incubate 3hr at 60C then 25hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed) | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 200ul (1ul | #Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | ||
#Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS | #Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS | ||
#Prepare RCA reaction mix | #Prepare RCA reaction mix '''on ice''' | ||
#*H2O - | #*H2O - 172ul | ||
#*Phi29 Buffer 10X - | #*Phi29 Buffer 10X - 20ul | ||
#*dNTP 25mM - | #*dNTP 25mM - 2ul | ||
#*aa-dUTP | #*aa-dUTP 2mM - 4ul | ||
#*Phi29 DNA polymerase 100U/ul - | #*Phi29 DNA polymerase 100U/ul - 2ul | ||
#Add RCA mix and incubate at 30C overnight (~14hrs) | #Add RCA mix and incubate at 30C overnight (~14hrs) | ||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add | #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 | #Wash with 1X PBS twice | ||
#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<!-- | ||
#Add 0.5uM FISGA_Adpt (1ul 100uM FISGA_Adpt + 199ul 2XSSC + 30% formamide) pre-heated to 75C and incubate 10min at RT | #Add 0.5uM FISGA_Adpt (1ul 100uM FISGA_Adpt + 199ul 2XSSC + 30% formamide) pre-heated to 75C and incubate 10min at RT | ||
#Wash with 2X SSC twice and add 1ml 2X SSC for imaging | #Wash with 2X SSC twice and add 1ml 2X SSC for imaging | ||
#Strip with 80% formamide pre-heated to 75C and incubate 15min at RT | #Strip with 80% formamide pre-heated to 75C and incubate 15min at RT | ||
#Wash with 1X PBS twice and store in 2ml PBS--> | #Wash with 1X PBS twice and store in 2ml PBS--> | ||
===Imaging=== | |||
*Continued onn [[Matt:LabNotes/2015-7-9]] |
Latest revision as of 02:11, 10 July 2015
Motor Neurons DARTFISH CA12kNov14_suppv2[edit]
- Picked up second batch iPS derived motor neurons from Yeo Lab (Sebastian)
- Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015
- RT for 15min
- Permeabilized/store in 70% EtOH at 4C (~72 hours before this DARTFISH experiment)
- Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015
- Last time forgot to add suppressor oligos
- 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[edit]
- Sample labeled "DARTFISH 6.19.2015"
- Wash the cells with 1ml of PBS (RNase free) 3 times
- Prepare RT mixture on ice
- DEPC-H2O - 157ul
- M-MuLV RT Buffer 10X - 20ul
- dNTP 25mM - 2ul
- aa-dUTP 2mM - 4ul
- 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 300ul 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
- Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
- Wash with 2ml H2O twice
- Prepare Ampligase mixture on ice
- Suppressor Oligos 50nM (labeled supp V4 ALL 5.18.15) - 55ul
- Ampligase Buffer 10X - 10ul
- Padlock Probes 438nM - 25ul
- Heat to 90C first and snap cool on ice block
- Ampligase 5U/ul - 10ul
- Add Ampligase mix to sample and incubate 3hr at 60C then 25hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed)
- Wash with 1X PBS once
- Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
- Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
- Prepare RCA reaction mix on ice
- H2O - 172ul
- Phi29 Buffer 10X - 20ul
- dNTP 25mM - 2ul
- aa-dUTP 2mM - 4ul
- Phi29 DNA polymerase 100U/ul - 2ul
- Add RCA mix and incubate at 30C overnight (~14hrs)
- 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
Imaging[edit]
- Continued onn Matt:LabNotes/2015-7-9