Matt:LabNotes/2016-4-28

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Test Diffusion of 5% PA 120um Gel Using CA12kNov2014_V4 Probes[edit]

Experiment Plan[edit]

  • Goal: Test whether the longer (150bp) padlock probes can diffuse through ~120um polyacrylamide hydrogel to generate rolonies
    • Previously PKP2_controlPP worked but in that experiment:
      • The concentration was 33X higher than in actual DARTFISH probeset (1nM vs 30pM)
      • PKP2_controlPP is half the length (~70bp vs 150bp)

Protocol[edit]

Day 1[edit]

  1. Prepare hydrogel mix
    1. 5% PA Gel: Standard hydrogel
  2. Attach ~120um thick adhesive to Vectabond + Bind-silane treated coverslip
  3. Add 6ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
    • Pipette away extra H2O leaving only dried beads
  4. Inject ~50ul of 200ul hydrogel mix into each dish (underneath another coverslip)
  5. Vacuum seal bag and then pump with Argon gas
  6. Let set at RT for 30min
  7. Attach to bottom of culture dish
  8. Wash with nf-H2O twice
  9. Prepare Ampligase mix on ice
    • Preheat H2O + Padlock probes to 85C and then snap cool before adding Ampligase buffer and enzyme
Component Volume
DEPC-H2O 52
Ampligase Buffer 10
Batch 15 368nM 28
Ampligase 10
Total 100
  1. Add mix to sample and incubate for 30min at 37C
  2. Move sample to 60C and oven slowly decreases to 55C and held for ~24hr

Day 2[edit]

  1. Wash with 1X PBS once
  2. Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 70C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. Add RCA mix and incubate 15hr at 30C
Component Volume
H2O 174
10X Phi29 Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
Phi 29 (low conc) 2
Total 200

Day 3[edit]

  1. Wash with 1X PBS once
  2. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris ph8.0 for 30min at RT
  5. Wash with 1X PBS twice and store in 2ml 1X PBS

Imaging[edit]

  • Used Gain and Laser power usually used for DARTFISH decoding
    • ch00: Cy5
    • ch01: Cy3
    • ch02: BF
  • Pos1: NegCtrl with no beads
    • No signal in any images, GOOD!
  • Pos2: Scattered beads
  • Pos3: Scattered beads

Before Hybridization[edit]

Hybridize FISGA_Adpt Cy3[edit]

  1. Preheat 200ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C
  2. Add 200ul to each dish and let sit for 10min at RT
  3. Wash twice with 2X SSC
  Cy3 Cy5
Pos 2 File:MAX Pos2 FISGA ch01.jpg File:MAX Pos2 FISGA ch00.jpg
Pos 3 File:MAX Pos3 FISGA ch01.jpg File:MAX Pos3 FISGA ch00.jpg

Strip[edit]

  1. Preheat 80% formamide 2X SSC to 75C
  2. Add 200ul to Dish1 (PosCtrl) and let sit for 15min at RT
  3. Wash twice with 1X PBS
  4. Add 500ul 2x SSC to image
  • Stripping of dye is incomplete
    • Need to wash more, strip longer, or make gel thinner so diffusion is faster
  • Some locations are stripped better than others

Hybridize Cycle5 Cy3 & Cy5[edit]

  1. Preheat 200ul 0.5uM Cycle5_Cy3 and 0.5uM Cycle5_Cy5 in 30% formamide 2X SSC to 75C
    • Expect Cy5 signal only, no Cy3
  2. Add 200ul to each dish and let sit for 10min at RT
  3. Wash twice with 2X SSC
  Cy3 Cy5
Pos 2 File:MAX Pos2 Cycle5 ch01.jpg File:MAX Pos2 Cycle5 ch00.jpg
Pos 3 File:MAX Pos3 Cycle5 ch01.jpg File:MAX Pos3 Cycle5 ch00.jpg
  • Specific Cy5 signal that wasn't there before
  • While most spots are on top of beads, they are definitely rolony specific because there are some spots that aren't near beads and these coincide with spots seen with FISGA_Cy3 dye

Conclusion[edit]

  • 140um 5% PA Gel is compatible with DARTFISH
  • However prevents complete stripping of dye with 80% formamide for 15min
    • Need to increase diffusion of stripped dyes (by making thinner gel)