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=Test Diffusion of PA Gel Formulations= *Using [[Matt:LabNotes/2016-3-31|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 ##Positive Control: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + 4ul of PKP2 beads ##5% PA Gel: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] ##5% PA Gel + 4% PFA: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + 4% PFA #Attach 3 ~120um thick adhesives to 3 Vectabond + Bind-silane treated coverslips #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 #Wash with nf-H2O twice #*5% PA Gel + 4% PFA failed to polymerize *Gels are sitting in 1X PBS at 4C #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||70 |- | Ampligase Buffer||10 |- | PKP2_controlPP 10nM||10 |- | 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 2=== #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 3=== #Wash with 1X PBS twice #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 ==Imaging== ====Hybridize FISGA_Adpt Cy3==== #Preheat 400ul 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 ====Stripped==== #Preheat 80% formamide to 75C and then add for 15min #Wash twice with 1X PBS ====Align Images and Subtract Stripped==== *Dish 1: PosCtrl [[File:Cropped_MAX_PosCtrl_BGsubtracted.jpg|450px]] *Dish 2: No Beads Area [[File:Cropped_MAX_5%25_NoBeads_BGsubtracted.jpg|450px]] *Dish 2: Beads [[File:Cropped_MAX_5%25_Beads_BGsubtracted.jpg|450px]] ==Conclusion== *33X concentration of padlock probes led to so much RCA product that there is no more punctate rolonies *Very few false positives in the "no beads" area *Next need to try if lower "real" concentration" with longer padlock probes (150bp) will also work
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