Matt:LabNotes/2015-7-20: Difference between revisions
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#*Suppressor Oligos 50nM (labeled supp V4 ALL 5.18.15) - 55ul | #*Suppressor Oligos 50nM (labeled supp V4 ALL 5.18.15) - 55ul | ||
#*Ampligase Buffer 10X - 10ul | #*Ampligase Buffer 10X - 10ul | ||
#*[[Matt: | #*[[Matt:JustinLabNotes/2015-7-6 | Padlock Probes 329nM]] - 38ul | ||
#**Heat to 90C first and snap cool on ice block | #**Heat to 90C first and snap cool on ice block | ||
#*Ampligase 5U/ul - 10ul | #*Ampligase 5U/ul - 10ul |
Revision as of 18:14, 21 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)
- 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 329nM - 38ul
- 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)
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