Matt:LabNotes/2015-7-20: Difference between revisions
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>Mzcai (Created page with "==Motor Neurons DARTFISH CA12kNov14_suppv2== *Picked up third batch iPS derived motor neurons from Yeo Lab (Sebastian) fixed on 7/17/2015 at 5pm **Fixed in 4% paraformaldehyde...") |
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=Motor Neurons Third Batch= | |||
*Picked up third batch iPS derived motor neurons from Yeo Lab (Sebastian) fixed on 7/17/2015 at 5pm | *Picked up third batch iPS derived motor neurons from Yeo Lab (Sebastian) fixed on 7/17/2015 at 5pm | ||
**Fixed in 4% paraformaldehyde RT for 15min | **Fixed in 4% paraformaldehyde RT for 15min | ||
**Permeabilized/store in 70% EtOH at 4C (~72 hours before | **Permeabilized/store in 70% EtOH at 4C (~72 hours before these experiments) | ||
==DARTFISH CA12kNov14_suppv2== | |||
*[[Matt:LabNotes/2015-6-19 | Repeat of second batch DARTFISH CA12kNov14_suppv2]] | *[[Matt:LabNotes/2015-6-19 | Repeat of second batch DARTFISH CA12kNov14_suppv2]] | ||
**Teach Justin how to do DARTFISH | **Teach Justin how to do DARTFISH | ||
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#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 | ||
Line 61: | Line 62: | ||
#Wash with 1X PBS twice and store in 2ml PBS--> | #Wash with 1X PBS twice and store in 2ml PBS--> | ||
== | |||
* | ==sm RNA FISH== | ||
*Using [[Matt:LabNotes/2015-7-14 | KIT-488 CUX2-594]] | |||
**Expect to see very few to no signals in FITC channel due to few KIT mRNA | |||
**If TXRED channel shows CUX2 transcripts then dye:probe=1.3 is not the problem | |||
**If TXRED channel shows bright regions then probe sequences not the problem | |||
*Using [[Matt:LabNotes/2015-7-16 | SNAP25-488 CUX2-647]] | |||
**Expect to see many SNAP25 spots in FITC channel | |||
**Hopefully can also see a couple CUX2 spots in Cy5 channel | |||
*[[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]] | |||
*[[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]] | |||
*Sample 1 labeled: "RNAFISH1 7.20.2015" | |||
**KIT-488 CUX2-594 | |||
*Sample 2 labeled: "RNAFISH2 7.20.2015" | |||
**SNAP25-488 CUX2-647 | |||
#Prepare 10ml Wash Buffer and let sit at RT | |||
#Thaw 300ul aliquot Hybridization Buffer and warm up to 37C | |||
#*Aliquots made by Dan | |||
#Prepare hybridization reaction | |||
#*Hybridization Buffer 100ul | |||
#*Probe 5ul + 5ul | |||
#Wash the cells with 1ml of PBS (RNase free) 2 times | |||
#Add 2ml Wash Buffer and let sit 10min at RT | |||
#Aspirate | |||
#Add 110ul hybridization solution to each sample and incubate overnight at 37C (~23hrs)<!-- | |||
#Prepare 15ml Wash Buffer | |||
#Warm up 12ml Wash Buffer and 3ml Wash Buffer with DAPI to 37C | |||
#Rinse with 2ml Wash Buffer | |||
#Add 2ml Wash Buffer and incubate 30min at 37C | |||
#Replace with 2ml 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 |
Revision as of 22:20, 20 July 2015
Motor Neurons Third Batch
- Picked up third batch iPS derived motor neurons from Yeo Lab (Sebastian) fixed on 7/17/2015 at 5pm
- Fixed in 4% paraformaldehyde RT for 15min
- Permeabilized/store in 70% EtOH at 4C (~72 hours before these experiments)
DARTFISH CA12kNov14_suppv2
- Repeat of second batch DARTFISH CA12kNov14_suppv2
- Teach Justin how to do DARTFISH
- May not decode these samples unless successful RNA FISH on this batch
- 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
DARTFISH with suppv2
- Sample labeled "DARTFISH 7.20.2015"
- 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)
sm RNA FISH
- Using KIT-488 CUX2-594
- Expect to see very few to no signals in FITC channel due to few KIT mRNA
- If TXRED channel shows CUX2 transcripts then dye:probe=1.3 is not the problem
- If TXRED channel shows bright regions then probe sequences not the problem
- Using SNAP25-488 CUX2-647
- Expect to see many SNAP25 spots in FITC channel
- Hopefully can also see a couple CUX2 spots in Cy5 channel
- Sample 1 labeled: "RNAFISH1 7.20.2015"
- KIT-488 CUX2-594
- Sample 2 labeled: "RNAFISH2 7.20.2015"
- SNAP25-488 CUX2-647
- Prepare 10ml Wash Buffer and let sit at RT
- Thaw 300ul aliquot Hybridization Buffer and warm up to 37C
- Aliquots made by Dan
- Prepare hybridization reaction
- Hybridization Buffer 100ul
- Probe 5ul + 5ul
- Wash the cells with 1ml of PBS (RNase free) 2 times
- Add 2ml Wash Buffer and let sit 10min at RT
- Aspirate
- Add 110ul hybridization solution to each sample and incubate overnight at 37C (~23hrs)
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