Matt:LabNotes/2015-7-20: Difference between revisions
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>Mzcai m (→Imaging) |
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#*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 (~18hrs) | #Transfer sample to 37C overnight (~18hrs) | ||
#Wash with 1X PBS once | #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 | #Add 300ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT | ||
Line 36: | Line 36: | ||
#Wash with 2ml H2O twice | #Wash with 2ml H2O twice | ||
#Prepare Ampligase mixture '''on ice''' | #Prepare Ampligase mixture '''on ice''' | ||
#*Suppressor Oligos | #*Suppressor Oligos 100nM (labeled supp V4 ALL 5.18.15) - 25.9ul | ||
#*Ampligase Buffer 10X - | #*Ampligase Buffer 10X - 9.1ul | ||
#*[[Matt: | #*[[Matt:JustinLabNotes/2015-7-6 | Padlock Probes 239nM]] - 38ul | ||
#**Heat to 90C first and snap cool on ice block | #**Heat to 90C first and snap cool on ice block | ||
#*Ampligase 5U/ul - | #*Ampligase 5U/ul - 9.1ul | ||
#*H2O - 7.9ul | |||
#*Total: 91ul | |||
#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) | #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) | ||
#*Incubated long time due to timing of starting RCA | |||
#*Middle of glass looked almost dry but edges were clearly wet | |||
#Wash with 1X PBS once | #Wash with 1X PBS once | ||
#Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | #Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C | ||
Line 51: | Line 55: | ||
#*aa-dUTP 2mM - 4ul | #*aa-dUTP 2mM - 4ul | ||
#*Phi29 DNA polymerase 100U/ul - 2ul | #*Phi29 DNA polymerase 100U/ul - 2ul | ||
#Add RCA mix and incubate at 30C overnight (~ | #Add RCA mix and incubate at 30C overnight (~16hrs) | ||
#Wash with 1X PBS once | #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 | #Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT | ||
Line 58: | Line 62: | ||
#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<!-- | ||
#Strip with 80% formamide pre-heated to 75C and incubate 15min at RT | #Strip with 80% formamide pre-heated to 75C and incubate 15min at RT | ||
#Wash with 1X PBS twice and store in 2ml PBS--> | #Wash with 1X PBS twice and store in 2ml PBS--> | ||
====Imaging==== | |||
*Check with Universal Rolony FISGA_Adpt | |||
*~30 rolonies per cell | |||
**Not great, could be better | |||
**High cell density | |||
*Pos1 | |||
[[File:MAX_20150723_DARTFISH_MN_FISGA_Pos1.jpg|450px]] | |||
*Pos2 | |||
[[File:MAX_20150723_DARTFISH_MN_FISGA_Pos2.jpg|450px]] | |||
==sm RNA FISH== | ==sm RNA FISH== | ||
Line 72: | Line 86: | ||
**Hopefully can also see a couple CUX2 spots in Cy5 channel | **Hopefully can also see a couple CUX2 spots in Cy5 channel | ||
===Procedure=== | |||
*[[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]] | ||
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#Add 2ml Wash Buffer and let sit 10min at RT | #Add 2ml Wash Buffer and let sit 10min at RT | ||
#Aspirate | #Aspirate | ||
#Add 110ul hybridization solution to each sample and incubate overnight at 37C (~23hrs) | #Add 110ul hybridization solution to each sample and incubate overnight at 37C (~23hrs) | ||
#Prepare | #Prepare 12ml Wash Buffer | ||
#Warm up | #Warm up 8ml Wash Buffer and 4ml Wash Buffer with DAPI to 37C | ||
#Rinse with 2ml Wash Buffer | #Rinse with 2ml Wash Buffer | ||
#Add 2ml Wash Buffer and incubate 30min at 37C | #Add 2ml Wash Buffer and incubate 30min at 37C | ||
Line 97: | Line 112: | ||
#Wash with 2X SSC (warmed to 30C) twice | #Wash with 2X SSC (warmed to 30C) twice | ||
#Add 2X SSC | #Add 2X SSC | ||
====Buffer Prep==== | ====Buffer Prep==== | ||
*Wash Buffer | *Wash Buffer | ||
Line 117: | Line 132: | ||
**RVC 200 mM (warm to 37) 100 uL | **RVC 200 mM (warm to 37) 100 uL | ||
**BSA 50 mg/mL 40 uL | **BSA 50 mg/mL 40 uL | ||
===Imaging=== | |||
====Zeiss Microscope in Huang Lab with Laser excitation==== | |||
*7/21/2015 | |||
*With help of Matt Walsh | |||
*Laser wavelengths: | |||
**488 | |||
**552? | |||
**642 | |||
*Exposure time: 100ms | |||
*Sample 1 - 488 (KIT) | |||
**Nothing to show, probably because gene expression is too low | |||
*Sample 1 - 594 (CUX2) | |||
**See combination of bright regions (guess is due to uncoupled dyes) and very few bright spots | |||
[[File:20150720_RNAFISH1_594.jpg]] | |||
*Sample 2 - 488 (SNAP25) | |||
**Lots of spots but also high background | |||
***Hard to say significantly more than what I have seen with CUX2 (though based on DARTFISH and FPKM it should be) | |||
[[File:20150720_RNAFISH2_488.jpg]] | |||
*Sample 2 - 647 (CUX2) | |||
**Image is very grainy making it hard to tell distinct spots from noise, probably needs better excitation parameters | |||
[[File:20150720_RNAFISH2_647.jpg]] | |||
====Zeiss Microscope in Huang Lab with Metal-Halide light==== | |||
*7/22/2015 |
Latest revision as of 02:11, 24 July 2015
Motor Neurons Third Batch[edit]
- 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[edit]
- 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[edit]
- 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 100nM (labeled supp V4 ALL 5.18.15) - 25.9ul
- Ampligase Buffer 10X - 9.1ul
- Padlock Probes 239nM - 38ul
- Heat to 90C first and snap cool on ice block
- Ampligase 5U/ul - 9.1ul
- H2O - 7.9ul
- Total: 91ul
- 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)
- Incubated long time due to timing of starting RCA
- Middle of glass looked almost dry but edges were clearly wet
- 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 - 172ul
- Phi29 Buffer 10X - 20ul
- dNTP 25mM - 2ul
- aa-dUTP 2mM - 4ul
- Phi29 DNA polymerase 100U/ul - 2ul
- Add RCA mix and incubate at 30C overnight (~16hrs)
- 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
- 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
Imaging[edit]
- Check with Universal Rolony FISGA_Adpt
- ~30 rolonies per cell
- Not great, could be better
- High cell density
- Pos1
File:MAX 20150723 DARTFISH MN FISGA Pos1.jpg
- Pos2
File:MAX 20150723 DARTFISH MN FISGA Pos2.jpg
sm RNA FISH[edit]
- 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
Procedure[edit]
- 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 12ml Wash Buffer
- Warm up 8ml Wash Buffer and 4ml 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[edit]
- 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
Imaging[edit]
Zeiss Microscope in Huang Lab with Laser excitation[edit]
- 7/21/2015
- With help of Matt Walsh
- Laser wavelengths:
- 488
- 552?
- 642
- Exposure time: 100ms
- Sample 1 - 488 (KIT)
- Nothing to show, probably because gene expression is too low
- Sample 1 - 594 (CUX2)
- See combination of bright regions (guess is due to uncoupled dyes) and very few bright spots
File:20150720 RNAFISH1 594.jpg
- Sample 2 - 488 (SNAP25)
- Lots of spots but also high background
- Hard to say significantly more than what I have seen with CUX2 (though based on DARTFISH and FPKM it should be)
- Lots of spots but also high background
File:20150720 RNAFISH2 488.jpg
- Sample 2 - 647 (CUX2)
- Image is very grainy making it hard to tell distinct spots from noise, probably needs better excitation parameters
File:20150720 RNAFISH2 647.jpg
Zeiss Microscope in Huang Lab with Metal-Halide light[edit]
- 7/22/2015