Matt:LabNotes/2015-8-4

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Revision as of 20:18, 6 August 2015 by >Mzcai (→‎DARTFISH on BA8)
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BA8 Section DARTFISH with Fiducial Beads

  • Practice DARTFISH on BA8 tissue sections
  • Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers

DARTFISH on BA8

  1. Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH sterilize and UV
  2. Made 40ml 4% PFA
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
  3. Take out BA8 section from -80C and incubate on 50C hot plate for 3min
  4. Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
    • Still had leak so used gap-filling adhesive 200 to seal
  7. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
    • 0.25% TX-100 in 2XSSPE tube had crack in cap so not completely sealed
  8. Wash with nuclease-free H2O three times
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with nuclease-free PBS three times
    • Can already see tissue is degrading
  11. Prepare Reverse Transcription Mix on ice
Components Volume
H2O 157
10X M-MuLV Buffer 20
25mM dNTP 2
2mM aa-dUTP 4
100uM N9 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~15hr at 37C
    • Tissue is slightly degraded but mostly okay
  2. Wash with 1X PBS once
  3. Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT
  4. Wash with 1X PBS twice
  5. Add Tris pH8.0 and incubate 30min at RT
  6. Wash with 1X PBS twice
  7. RNA Removal
    • Add RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  8. Wash with H2O twice
  9. Add 66nM Ampligase Mix (no suppressor) and incubate ~3hr at 60C as oven slowly decreases to 55C and held for another ~16hr
    • Wrapped with parafilm to prevent evaporation (works very well)
Component Volume
DEPC-H2O 121
Ampligase Buffer 20
Matt:JustinLabNotes/2015-7-9#Qubit_Quantification 39
Ampligase 20
Total 200
  1. Wash with 1X PBS once
  2. Add 1uM FISSEQ_ppRCA (0.5ul 200uM FISSEQ_ppRCA + 199.5ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 65C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. Add RCA mix and incubate __hr at 30C
Component Volume
H2O 172
10X Phi29 Buffer 20
25mM dNTP 2
2mM aa-dUTP 4
Phi 29 (low conc) 2
Total 200