Matt:LabNotes/2015-7-20: Difference between revisions

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==Motor Neurons DARTFISH CA12kNov14_suppv2==
=Motor Neurons Third Batch=
*Picked up third batch iPS derived motor neurons from Yeo Lab (Sebastian) fixed on 7/17/2015 at 5pm
*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
**Fixed in 4% paraformaldehyde RT for 15min
**Permeabilized/store in 70% EtOH at 4C (~72 hours before this DARTFISH experiment)
**Permeabilized/store in 70% EtOH at 4C (~72 hours before these experiments)


==DARTFISH CA12kNov14_suppv2==
*[[Matt:LabNotes/2015-6-19 | Repeat of second batch DARTFISH CA12kNov14_suppv2]]
*[[Matt:LabNotes/2015-6-19 | Repeat of second batch DARTFISH CA12kNov14_suppv2]]
**Teach Justin how to do DARTFISH
**Teach Justin how to do DARTFISH
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#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
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#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]]
==sm RNA FISH==
*Using [[Matt:LabNotes/2015-7-14 | 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 [[Matt:LabNotes/2015-7-16 | SNAP25-488 CUX2-647]]
**Expect to see many SNAP25 spots in FITC channel
**Hopefully can also see a couple CUX2 spots in Cy5 channel
 
*[[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]]
*[[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]]
 
*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 15ml Wash Buffer
#Warm up 12ml Wash Buffer and 3ml 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====
*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

Revision as of 22:20, 20 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

  • 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"
  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)


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
  1. Prepare 10ml Wash Buffer and let sit at RT
  2. Thaw 300ul aliquot Hybridization Buffer and warm up to 37C
    • Aliquots made by Dan
  3. Prepare hybridization reaction
    • Hybridization Buffer 100ul
    • Probe 5ul + 5ul
  4. Wash the cells with 1ml of PBS (RNase free) 2 times
  5. Add 2ml Wash Buffer and let sit 10min at RT
  6. Aspirate
  7. 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