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=Test 25um Gel= *[[Matt:LabNotes/2016-4-28|Like this test but thinner gel]] **Use Kapton tape to make thinner gel *Using [[Matt:LabNotes/2016-3-31|PKP2 Beads]] as targets ==Experiment Plan== *Goal: Test whether the longer (150bp) padlock probes can diffuse through ~25-50um polyacrylamide hydrogel to generate rolonies **Previously worked with ~140um gel ==Protocol== ===Day 1=== #Prepare hydrogel mix ##5% PA Gel: [[Matt:LabNotes/2016-2-9#Experiment|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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''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=== #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 {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||174 |- | 10X Phi29 Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | Phi 29 (low conc)||2 |- | Total||200 |} ===Day 4=== #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== *8-bit to save space *20X Objective for larger field of view ====Hybridize FISGA_Adpt Cy3==== #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|450px]] ====Strip==== #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|450px]] ==Conclusion== *25-50um (Kapton tape thickness) 5% PA Gel is thin enough that a single round of stripping/washing completely removes signal
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