Matt:LabNotes/2016-5-16
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Test 25um Gel[edit]
- Like this test but thinner gel
- Use Kapton tape to make thinner gel
- Using PKP2 Beads as targets
Experiment Plan[edit]
- Goal: Test whether the longer (150bp) padlock probes can diffuse through ~25-50um polyacrylamide hydrogel to generate rolonies
- Previously worked with ~140um gel
Protocol[edit]
Day 1[edit]
- Prepare hydrogel mix
- 5% PA Gel: Standard hydrogel
- Attach ~25um thick adhesive to Vectabond + Bind-silane treated coverslip
- Add 2ul 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
Day 2[edit]
- 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 | 37 |
Ampligase Buffer | 10 |
Batch 14 237nM | 43 |
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 3[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 4[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]
- 8-bit to save space
- 20X Objective for larger field of view
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
File:MAX Beads FISGAadpt Cy3.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 1X PBS to image
File:MAX Beads 15min 2xWash.jpg
Conclusion[edit]
- 25-50um (Kapton tape thickness) 5% PA Gel is thin enough that a single round of stripping/washing completely removes signal