Matt:LabNotes/2015-6-19: Difference between revisions

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==Motor Neurons DARTFISH CA12kNov14_suppv2==
*[[Matt:LabNotes/2015-6-18 | Picked up second batch iPS derived motor neurons from Yeo Lab (Sebastian)]]
**Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015
***RT for 15min
**Permeabilized/store in 70% EtOH at 4C (~72 hours before this DARTFISH experiment)
*[[Matt:LabNotes/2015-6-8#DARTFISH | Last time forgot to add suppressor oligos]]
**Use 1000X concentration of suppressor oligos to match [[Matt:LabNotes/2015-5-18 | in vitro experiment]]
***e.g. 100nM CA12kNov2014_V4 = 28.5pM per padlock probe, therefore 28.5nM of each suppressor oligo
===DARTFISH with suppv2===
===DARTFISH with suppv2===
*Sample: 37C 10min
*Sample labeled "DARTFISH 6.19.2015"
**Labeled "DARTFISH 6.19.2015"


#Wash the cells with 1ml of PBS (RNase free) 3 times
#Wash the cells with 1ml of PBS (RNase free) 3 times
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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 (~15hrs)<!--
#Transfer sample to 37C overnight (~18hrs)
#Wash with 1X PBS once
#Wash with 1X PBS once
#Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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
#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
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#Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
#Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
#Wash with 2ml H2O twice
#Wash with 2ml H2O twice
#Prepare Ampligase mixture (2X volume) '''on ice'''
#Prepare Ampligase mixture '''on ice'''
#*H2O - 110ul
#*Suppressor Oligos 50nM (labeled supp V4 ALL 5.18.15) - 55ul
#*Ampligase Buffer 10X - 20ul
#*Ampligase Buffer 10X - 10ul
#*[[Matt:LabNotes/2015-5-11#Qubit_Quantification | Padlock Probes 461nM]] - 50ul
#*[[Matt:LabNotes/2015-6-3#Qubit_ssDNA_Quantification | Padlock Probes 438nM]] - 25ul
#**Heat to 90C first and snap cool on ice block
#**Heat to 90C first and snap cool on ice block
#*Ampligase 5U/ul - 20ul
#*Ampligase 5U/ul - 10ul
#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)
#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)
#*3:20p-3p
#Wash with 1X PBS once
#Wash with 1X PBS once
#Add 200ul (1ul 100uM 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
#Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
#Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
#Prepare RCA reaction mix (2X volume) '''on ice'''
#Prepare RCA reaction mix '''on ice'''
#*H2O - 348ul
#*H2O - 172ul
#*Phi29 Buffer 10X - 40ul
#*Phi29 Buffer 10X - 20ul
#*dNTP 25mM - 4ul
#*dNTP 25mM - 2ul
#*aa-dUTP 4mM - 4ul
#*aa-dUTP 2mM - 4ul
#*Phi29 DNA polymerase 100U/ul - 4ul
#*Phi29 DNA polymerase 100U/ul - 2ul
#Add RCA mix and incubate at 30C overnight (~14hrs)
#Add RCA mix and incubate at 30C overnight (~14hrs)
#Wash with 1X PBS once
#Wash with 1X PBS once
#Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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
#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
#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===
*Continued onn [[Matt:LabNotes/2015-7-9]]

Latest revision as of 02:11, 10 July 2015

Motor Neurons DARTFISH CA12kNov14_suppv2[edit]

DARTFISH with suppv2[edit]

  • Sample labeled "DARTFISH 6.19.2015"
  1. Wash the cells with 1ml of PBS (RNase free) 3 times
  2. 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
  3. Add RT mixture and incubate for 10min at 4C
  4. Transfer sample to 37C overnight (~18hrs)
  5. Wash with 1X PBS once
  6. Add 300ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  7. Wash with 1X PBS twice
  8. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  9. Wash with 1X PBS twice
  10. Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
  11. Wash with 2ml H2O twice
  12. Prepare Ampligase mixture on ice
    • Suppressor Oligos 50nM (labeled supp V4 ALL 5.18.15) - 55ul
    • Ampligase Buffer 10X - 10ul
    • Padlock Probes 438nM - 25ul
      • Heat to 90C first and snap cool on ice block
    • Ampligase 5U/ul - 10ul
  13. 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)
  14. Wash with 1X PBS once
  15. Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
  16. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  17. 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
  18. Add RCA mix and incubate at 30C overnight (~14hrs)
  19. Wash with 1X PBS once
  20. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  21. Wash with 1X PBS twice
  22. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  23. Wash with 1X PBS twice

Imaging[edit]