Matt:LabNotes/2016-4-28
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Test Diffusion of 5% PA 120um Gel Using CA12kNov2014_V4 Probes
- Using PKP2 Beads as targets
- PKP2_controlPP
Dilute PKP2_controlPP
- Resuspended to 10uM with 450ul H2O
- Serial dilute to 10nM
- 10:1 dilution 3 times
Experiment Plan
- Goal: Test whether padlock probes and polymerases can diffuse through ~100um? polyacrylamide hydrogel to generate rolonies
- Dish1: Positive control: mix beads into hydrogel solution before polymerization
- This way some beads will be close to the surface, minimizing distance molecules need to diffuse
- Can't do it without hydrogel matrix because the beads and rolonies need some sort of substrate
- Dish2: 5% Polyacrylamide Gel (199:1 Acrylamide:Bis-acrylamide)
- Beads are underneath gel, being held in place by magnets
- Do padlock probe capture and then RCA to generate rolonies in hydrogel matrix
Protocol
Day 1
- 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