Matt:LabNotes/2016-5-16
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Test 140um Gel
- Like this test but thinner gel
- Using PKP2 Beads as targets
Experiment Plan
- 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
Day 1
- 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
- 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