Matt:LabNotes/2015-7-1: Difference between revisions

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(Created page with "==Motor Neurons DARTFISH CA12kNov14_suppv2== *Received Bipolar Neurons (iNGN) from Alex at Harvard **Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015 ***RT for 15min **...")
 
>Mzcai
 
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==Motor Neurons DARTFISH CA12kNov14_suppv2==
==Motor Neurons DARTFISH CA12kNov14_suppv2==
*Received Bipolar Neurons (iNGN) from Alex at Harvard
*Received Bipolar Neurons (iNGN) from Alex at Harvard
**Fixed in 4% paraformaldehyde (fresh) at 3:00p 6/16/2015
**Fixed in 4% paraformaldehyde (fresh) 6/30/2015
***RT for 15min
***RT for 15min
**Permeabilized/store in 70% EtOH at 4C (~24 hours before this DARTFISH experiment)
**Permeabilized/store in 70% EtOH at 4C (~24 hours before this DARTFISH experiment)
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===DARTFISH with suppv2===
===DARTFISH with suppv2===
*Sample labeled "DARTFISH 7.1.2015"
====Sample labeled "DARTFISH 7.1.2015"====
#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
#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"====
*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
#Wash the cells with 1ml of PBS (RNase free) 3 times
#Prepare RT mixture '''on ice'''
#Prepare RT mixture '''on ice'''
#*DEPC-H2O - 157ul
#*DEPC-H2O - 159ul
#*M-MuLV RT Buffer 10X - 20ul
#*M-MuLV RT Buffer 10X - 20ul
#*dNTP 25mM - 2ul
#*dNTP 25mM - 2ul
#*aa-dUTP 2mM - 4ul
#*aa-dUTP 4mM - 2ul
#*RT primer 100uM - 5ul
#*RT primer 100uM - 5ul
#**Nonamer
#**Nonamer
Line 23: Line 46:
#*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 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
#Store at 4C
====Continued protocol with DARTFISH2====
*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
#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 '''on ice'''
#Prepare Ampligase mixture '''on ice'''
#*Suppressor Oligos 50nM (labeled supp V4 ALL 5.18.15) - 55ul
#*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
#*Ampligase Buffer 10X - 10ul
#*[[Matt:LabNotes/2015-6-3#Qubit_ssDNA_Quantification | Padlock Probes 438nM]] - 25ul
#*[[Matt:LabNotes/2015-6-29#Qubit_Quantification | Padlock Probes 227nM]] - 38ul (should be 44ul for 100nM total)
#**Heat to 90C first and snap cool on ice block
#**Heat to 90C first and snap cool on ice block (FORGOT TO HEAT)
#*H2O - 17ul
#*Ampligase 5U/ul - 10ul
#*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)
#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
#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
#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 '''on ice'''
#Prepare RCA reaction mix '''on ice'''
#*H2O - 172ul
#*H2O - 174ul
#*Phi29 Buffer 10X - 20ul
#*Phi29 Buffer 10X - 20ul
#*dNTP 25mM - 2ul
#*dNTP 25mM - 2ul
#*aa-dUTP 2mM - 4ul
#*aa-dUTP 4mM - 2ul
#*Phi29 DNA polymerase 100U/ul - 2ul
#*Phi29 DNA polymerase 100U/ul - 2ul
#Add RCA mix and incubate at 30C overnight (~14hrs)
#Add RCA mix and incubate at 30C overnight (~18hrs)
#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
#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 and store at 4C<!--
#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 on [[Matt:LabNotes/2015-7-9]]

Latest revision as of 02:12, 10 July 2015

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)

DARTFISH with suppv2[edit]

Sample labeled "DARTFISH 7.1.2015"[edit]

  1. Wash the cells with 1ml of PBS (RNase free) 3 times
  2. 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
  3. Add RT mixture and incubate for 10min at 4C
  4. 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
  5. Wash with 1X PBS once
  6. Add 200ul 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. 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
  1. Wash the cells with 1ml of PBS (RNase free) 3 times
  2. 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
  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 200ul 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. Store at 4C

Continued protocol with DARTFISH2[edit]

  • Only had enough padlock probes for 1 dish so chose DARTFISH2 7.2.2015
  1. Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C
  2. Wash with 2ml H2O twice
  3. 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
  4. 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?
  5. Wash with 1X PBS once
  6. Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
  7. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  8. 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
  9. Add RCA mix and incubate at 30C overnight (~18hrs)
  10. Wash with 1X PBS once
  11. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT
  12. Wash with 1X PBS twice
  13. Add 200ul 1M Tris (pH 8.0) for 30min at RT
  14. Wash with 1X PBS twice and store at 4C

Imaging[edit]