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===Day 1=== #Prepare hydrogel mix ##5% PA Gel: [[Matt:LabNotes/2016-2-9#Experiment|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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''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
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