Matt:LabNotes/2015-6-8: Difference between revisions
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>Mzcai (Created page with "===RNA FISH=== *From SLC6A1-488 SATB2-594 Probes * Dan's best practice FISH protocol *[[Daniel...") |
>Mzcai m (→RNA FISH) |
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==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 | |||
===RNA FISH=== | ===RNA FISH=== | ||
* | *Using [[Matt:LabNotes/2015-6-5#Dye_Coupling | SLC6A1-488 SATB2-594 Probes]] | ||
*[[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]] | *[[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]] | ||
*[[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]] | *[[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]] | ||
*Sample: RT 15min | |||
**Labeled "RNAFISH1 6.8.2015" | |||
*Sample: 37C 10min | |||
**Labeled "RNAFISH2 6.8.2015" | |||
#Prepare 8ml Wash Buffer and let sit at RT | |||
#Thaw 500ul aliquot Hybridization Buffer and warm up to 37C | |||
#*Aliquots made by Dan | |||
#Prepare hybridization reaction | |||
#*Hybridization Buffer 200ul | |||
#*Probe | |||
#Wash the cells with 1ml of PBS (RNase free) 2 times | |||
#Add hybridization solution to sample and incubate overnight at 37C (7p-) | |||
====Buffer Prep==== | |||
*Wash Buffer | |||
**20X SSC 5mL | |||
**Formamide 5mL | |||
**RNase free H2O 40ml | |||
*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 | |||
#*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 (7pm-) | |||
===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 (7pm-) |
Revision as of 01:08, 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 8ml Wash Buffer and let sit at RT
- Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
- Aliquots made by Dan
- Prepare hybridization reaction
- Hybridization Buffer 200ul
- Probe
- Wash the cells with 1ml of PBS (RNase free) 2 times
- Add hybridization solution to sample and incubate overnight at 37C (7p-)
Buffer Prep
- Wash Buffer
- 20X SSC 5mL
- Formamide 5mL
- RNase free H2O 40ml
- 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
- 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 (7pm-)
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 (7pm-)