Matt:LabNotes/2015-7-1
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Motor Neurons DARTFISH CA12kNov14_suppv2[edit]
- Received Bipolar Neurons (iNGN) from Alex at Harvard
- Fixed in 4% paraformaldehyde (fresh) 6/30/2015
- RT for 15min
- Permeabilized/store in 70% EtOH at 4C (~24 hours before this DARTFISH experiment)
- Fixed in 4% paraformaldehyde (fresh) 6/30/2015
- Follow protocol from last time
- 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 7.1.2015"[edit]
- 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
- 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)
- Center of glass was dry and there was solution around edges of dish
- The vortexer was shaking all night in the incubator and the vibration may disturb the solution
- Center of glass was dry and there was solution around edges of dish
- 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
- Store at 4C
Sample labeled "DARTFISH2 7.2.2015"[edit]
- Because first sample had dry areas after RT preparing a second sample just in case
- 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
- 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 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
- Store at 4C
Continued protocol with DARTFISH2[edit]
- Only had enough padlock probes for 1 dish so chose DARTFISH2 7.2.2015
- 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 100nM (labeled V4 supp v2 100nm each ALL (40+5) 7.6.15) - 25ul
- 10ul V4 5supp 10uM each + 100ul V4 40supp 1uM each + 890ul H2O = 1000ul 100nM each
- Ampligase Buffer 10X - 10ul
- Padlock Probes 227nM - 38ul (should be 44ul for 100nM total)
- Heat to 90C first and snap cool on ice block (FORGOT TO HEAT)
- H2O - 17ul
- Ampligase 5U/ul - 10ul
- Suppressor Oligos 100nM (labeled V4 supp v2 100nm each ALL (40+5) 7.6.15) - 25ul
- Add Ampligase mix to sample and incubate 3hr at 60C then 21hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed)
- Middle of glass was dry?
- 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 - 174ul
- Phi29 Buffer 10X - 20ul
- dNTP 25mM - 2ul
- aa-dUTP 4mM - 2ul
- Phi29 DNA polymerase 100U/ul - 2ul
- Add RCA mix and incubate at 30C overnight (~18hrs)
- 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 and store at 4C
Imaging[edit]
- Continued on Matt:LabNotes/2015-7-9