Matt:LabNotes/2016-4-28
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Test Diffusion of 5% PA 120um Gel Using CA12kNov2014_V4 Probes[edit]
- Using PKP2 Beads as targets
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)
- Previously PKP2_controlPP worked but in that experiment:
Protocol[edit]
Day 1[edit]
- Prepare hydrogel mix
- 5% PA Gel: Standard hydrogel
- Attach ~120um thick adhesive to Vectabond + Bind-silane treated coverslip
- Add 6ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
- Pipette away extra H2O leaving only dried beads
- Inject ~50ul of 200ul hydrogel mix into each dish (underneath another coverslip)
- Vacuum seal bag and then pump with Argon gas
- Let set at RT for 30min
- Attach to bottom of culture dish
- Wash with nf-H2O twice
- 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 |
- Add mix to sample and incubate for 30min at 37C
- Move sample to 60C and oven slowly decreases to 55C and held for ~24hr
Day 2[edit]
- Wash with 1X PBS once
- Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
- Preheated to 70C before adding
- Wash with 2X SSC once, 1X SSC once, and 1X PBS once
- 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]
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT
- Wash with 1X PBS twice
- Add 1M Tris ph8.0 for 30min at RT
- 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]
- No fluorescent signal anywhere
- Pos 2: File:Pos2 Before z05 ch02.jpg
- Pos 3: File:Pos3 Before z06 ch02.jpg
Hybridize FISGA_Adpt Cy3[edit]
- Preheat 200ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C
- Add 200ul to each dish and let sit for 10min at RT
- 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]
- Preheat 80% formamide 2X SSC to 75C
- Add 200ul to Dish1 (PosCtrl) and let sit for 15min at RT
- Wash twice with 1X PBS
- 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]
- Preheat 200ul 0.5uM Cycle5_Cy3 and 0.5uM Cycle5_Cy5 in 30% formamide 2X SSC to 75C
- Expect Cy5 signal only, no Cy3
- Add 200ul to each dish and let sit for 10min at RT
- 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)