Matt:LabNotes/2015-6-8: Difference between revisions
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>Mzcai m (→RNA FISH) |
>Mzcai mNo edit summary |
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#Add Wash Buffer and let sit 10min at RT | #Add Wash Buffer and let sit 10min at RT | ||
#Aspirate | #Aspirate | ||
#Add 110ul hybridization solution to each sample and incubate overnight at 37C ( | #Add 110ul hybridization solution to each sample and incubate overnight at 37C (~18hrs) | ||
#Warm up 6ml Wash Buffer and 2ml Wash Buffer with DAPI to 37C | |||
#Rinse with 2ml Wash Buffer | |||
#Add 1ml Wash Buffer and incubate 30min at 37C | |||
#Replace with 1ml Wash Buffer with DAPI and incubate 30min at 30C | |||
#Wash with 2X SSC (warmed to 30C) twice | |||
#Add 2X SSC | |||
====Buffer Prep==== | ====Buffer Prep==== | ||
Line 35: | Line 41: | ||
**Formamide 5mL | **Formamide 5mL | ||
**RNase free H2O 40ml | **RNase free H2O 40ml | ||
*Wash Buffer with DAPI | |||
**Wash Buffer 10ml | |||
**DAPI (100ug/ml) 50ul | |||
***Stored in brown opaque tube in 4C | |||
*Hybridization Buffer | *Hybridization Buffer | ||
Line 60: | Line 71: | ||
#*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 (~15hrs) | ||
#Wash with 1X PBS once | |||
#Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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 CircLigase mixture '''on ice''' | |||
#*H2O - 128ul | |||
#*CircLigase Buffer 10X - 20ul | |||
#*MnCl2 50mM - 10ul | |||
#*Betaine 5M - 40ul | |||
#*CircLigase II 100U/ul - 2ul | |||
#Add CircLigase mix to sample and incubate 2hr at 60C | |||
#Wash with 1X PBS twice | |||
===DARTFISH=== | ===DARTFISH=== | ||
Line 79: | Line 105: | ||
#*M-MuLV reverse transcriptase 100U/ul - 20ul | #*M-MuLV reverse transcriptase 100U/ul - 20ul | ||
#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 (~15hrs) | ||
#Wash with 1X PBS once | |||
#Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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 (2X volume)'''on ice''' | |||
#*H2O - ul | |||
#*Ampligase Buffer 10X - 20ul | |||
#*Padlock Probes - ul | |||
#*Ampligase 5U/ul - 20ul | |||
#Add Ampligase mix to sample and incubate 2hr at 60C then 22hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed) |
Revision as of 19:40, 9 June 2015
RNA in situ Differentiated Motor Neurons
- Picking up iPS derived motor neurons from Yeo Lab (Sebastian)
- Fixed in 4% paraformaldehyde (fresh) at 8:30a 6/8/2015
- 4 at RT for 10min
- 8 at RT for 15min
- 8 at 37C for 10min
- Permeabilized/store in 70% EtOH at -20C for ~5hr
- After bringing to our lab put in 4C > 3hr
- Fixed in 4% paraformaldehyde (fresh) at 8:30a 6/8/2015
RNA FISH
- Sample: RT 15min
- Labeled "RNAFISH1 6.8.2015"
- Sample: 37C 10min
- Labeled "RNAFISH2 6.8.2015"
- Prepare 10ml Wash Buffer and let sit at RT
- Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
- Aliquots made by Dan
- Prepare hybridization reaction (2X volume)
- Hybridization Buffer 200ul
- Probe 10ul of each (488 and 594)
- Wash the cells with 1ml of PBS (RNase free) 2 times
- Add Wash Buffer and let sit 10min at RT
- Aspirate
- Add 110ul hybridization solution to each sample and incubate overnight at 37C (~18hrs)
- Warm up 6ml Wash Buffer and 2ml Wash Buffer with DAPI to 37C
- Rinse with 2ml Wash Buffer
- Add 1ml Wash Buffer and incubate 30min at 37C
- Replace with 1ml Wash Buffer with DAPI and incubate 30min at 30C
- Wash with 2X SSC (warmed to 30C) twice
- Add 2X SSC
Buffer Prep
- Wash Buffer
- 20X SSC 5mL
- Formamide 5mL
- RNase free H2O 40ml
- Wash Buffer with DAPI
- Wash Buffer 10ml
- DAPI (100ug/ml) 50ul
- Stored in brown opaque tube in 4C
- Hybridization Buffer
- RNAse free water 5.3 mL
- SSC 20X 1 mL
- Dextran sulfate 2 mL
- Formamide 1 mL
- E coli tRNA 500 uL
- RVC 200 mM (warm to 37) 100 uL
- BSA 50 mg/mL 40 uL
FISSEQ
- Sample: RT 15min
- Labeled "FISSEQ +ctrl 6.8.2015"
- 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
- FISSEQ_RT primer 100uM - 5ul
- /5Phos/TCTCGGGAACGCTGAAGANNNNNN
- 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 (~15hrs)
- Wash with 1X PBS once
- Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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 CircLigase mixture on ice
- H2O - 128ul
- CircLigase Buffer 10X - 20ul
- MnCl2 50mM - 10ul
- Betaine 5M - 40ul
- CircLigase II 100U/ul - 2ul
- Add CircLigase mix to sample and incubate 2hr at 60C
- Wash with 1X PBS twice
DARTFISH
- Sample: RT 15min
- Labeled "DARTFISH1 6.8.2015"
- Sample: 37C 10min
- Labeled "DARTFISH2 6.8.2015"
- Wash the cells with 1ml of PBS (RNase free) 3 times
- Prepare RT mixture on ice
- DEPC-H2O - 318ul
- M-MuLV RT Buffer 10X - 40ul
- dNTP 25mM - 4ul
- aa-dUTP 4mM - 4ul
- RT primer 100uM - 10ul
- Nonamer
- RNase inhibitor 40U/ul - 4ul
- M-MuLV reverse transcriptase 100U/ul - 20ul
- Add RT mixture and incubate for 10min at 4C
- Transfer sample to 37C overnight (~15hrs)
- Wash with 1X PBS once
- Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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 (2X volume)on ice
- H2O - ul
- Ampligase Buffer 10X - 20ul
- Padlock Probes - ul
- Ampligase 5U/ul - 20ul
- Add Ampligase mix to sample and incubate 2hr at 60C then 22hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed)