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=Test Diffusion of 5% PA 120um Gel Using CA12kNov2014_V4 Probes= *Using [[Matt:LabNotes/2016-3-31|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) ==Protocol== ===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 ===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 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== *Used Gain and Laser power usually used for DARTFISH decoding **ch00: Cy5 **ch01: Cy3 **ch02: BF *Pos1: NegCtrl with no beads **No signal in any images, GOOD! *Pos2: Scattered beads *Pos3: Scattered beads ====Before Hybridization==== *No fluorescent signal anywhere *Pos 2: [[File:Pos2_Before_z05_ch02.jpg|450px]] *Pos 3: [[File:Pos3_Before_z06_ch02.jpg|450px]] ====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 {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | align="right" width="51" height="14" | | width="51" | Cy3 | width="51" | Cy5 |- style="font-size:11pt" valign="bottom" | height="14" | Pos 2 | [[File:MAX_Pos2_FISGA_ch01.jpg|450px]] | [[File:MAX_Pos2_FISGA_ch00.jpg|450px]] |- style="font-size:11pt" valign="bottom" | height="14" | Pos 3 | [[File:MAX_Pos3_FISGA_ch01.jpg|450px]] | [[File:MAX_Pos3_FISGA_ch00.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 2x SSC to image *Pos 2: [[File:MAX_Pos2_Stripped_ch01.jpg|450px]] *Pos 3: [[File:MAX_Pos3_Stripped_ch01.jpg|450px]] *Stripping of dye is incomplete **Need to wash more, strip longer, or make gel thinner so diffusion is faster *Some locations are stripped better than others ====Hybridize Cycle5 Cy3 & Cy5==== #Preheat 200ul 0.5uM Cycle5_Cy3 and 0.5uM Cycle5_Cy5 in 30% formamide 2X SSC to 75C #*Expect Cy5 signal only, no Cy3 #Add 200ul to each dish and let sit for 10min at RT #Wash twice with 2X SSC {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | align="right" width="51" height="14" | | width="51" | Cy3 | width="51" | Cy5 |- style="font-size:11pt" valign="bottom" | height="14" | Pos 2 | [[File:MAX_Pos2_Cycle5_ch01.jpg|450px]] | [[File:MAX_Pos2_Cycle5_ch00.jpg|450px]] |- style="font-size:11pt" valign="bottom" | height="14" | Pos 3 | [[File:MAX_Pos3_Cycle5_ch01.jpg|450px]] | [[File:MAX_Pos3_Cycle5_ch00.jpg|450px]] |} *Specific Cy5 signal that wasn't there before *While most spots are on top of beads, they are definitely rolony specific because there are some spots that aren't near beads and these coincide with spots seen with FISGA_Cy3 dye ==Conclusion== *140um 5% PA Gel is compatible with DARTFISH *However prevents complete stripping of dye with 80% formamide for 15min **Need to increase diffusion of stripped dyes (by making thinner gel)
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