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=Test Diffusion of PA Gel Formulations= *Using [[Matt:LabNotes/2016-3-31|PKP2 Beads]] as targets *CA12kNov2014 V4 Padlock Probes ==Experiment Plan== *Goal: Test whether padlock probes and polymerases can diffuse through ~400um 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: 10% Polyacrylamide Gel (199:1 Acrylamide:Bis-acrylamide) **Beads are underneath gel, being held in place by magnets *Dish3: 10% Polyacrylamide (199:1) + 4% PFA Gel **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]] + 2ul of PKP2 beads ##10% PA Gel: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] ##10% PA Gel + 4% PFA: [[Matt:LabNotes/2016-2-9#Experiment|Standard hydrogel]] + 4% PFA #Attach 3 Vectabond + Bind-silane treated coverslips to 3 culture dishes #Add 3ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets #*Pipette away extra H2O leaving only dried beads #*[[File:20160411_magnetbeads_culturedish.jpg|250px]] #Inject ~70ul of 200ul hydrogel mix into each dish (underneath the plastic insert) #Vacuum seal bag and then pump with Argon gas #Let set at RT for 30min #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 *Combine B11 and B12: 100ul at ~200nM **B12 looked to have some very fine PA gel still in there (not filtered enough?) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | DEPC-H2O||47 |- | Ampligase Buffer||10 |- | Batch 11 & 12 Padlock Probes ~200nM]]||33 |- | 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=== *Dish1 showed signs of leaking but the gel was not dried out and there was still some liquid on top **Later confirmed to definitely be leaking #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== ===Dish 1: Positive Control=== *Beads well dispersed among many z layers **No z-slice has them all in focus ====Hybridize FISGA_Adpt Cy3==== #Preheat 600ul 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 *Position 1 **Initially thought good, there is signal as expected [[File:MAX_Dish1_PosCtrl_FISGA_Adpt_Pos1.jpg|450px]] *Position 2 **See some spots but they overlap with bead position (and not the beads closest to the top surface) **Not consistent with position 1 results [[File:Dish1_PosCtrl_FISGA_Adpt_Pos2_z07_ch01.jpg|450px]][[File:MAX_Dish1_PosCtrl_FISGA_Adpt_Pos2.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 *Bumped dish, lost position but found again *Position 1 (Cy3) **Not good sign, signal is still there... even though it looks like less [[File:MAX_Dish1_PosCtrl_Stripped_Cy3_Pos1.jpg|450px]] *Position 1 (Cy5) [[File:MAX_Dish1_PosCtrl_Stripped_Cy5_Pos1.jpg|450px]] ====Hybridize Cycle5 Cy3 & Cy5==== *Only Cy5 should show signal *Position 1 (Cy3) [[File:MAX_Dish1_PosCtrl_Cycle5_Cy3_Pos1.jpg|450px]] *Position 1 (Cy5) **Good there's signal when there wasn't before! [[File:MAX_Dish1_PosCtrl_Cycle5_Cy5_Pos1.jpg|450px]] ===Dish 2: 10% PA=== *Beads are clearly in focus in single z-plane **High density where magnet was [[File:Dish2_10%25PA_z07_ch01.jpg|450px]] *Hybridized FISGA_Adpt_Cy3 but only a few fluorescent spots, not sure if rolonies or beads... **Turned up brightness in ImageJ and can see dim beads **However if the few bright spots are rolonies I expected to see more [[File:MAX_Dish2_10%25PA.jpg|450px]] ===Dish 3: 10% PA 4% PFA=== *Beads are clearly in focus in single z-plane **High density where magnet was [[File:Dish3_10%25PA_4%25PFA_z07_ch01.jpg|450px]] *Hybridized FISGA_Adpt_Cy3 but No Fluorescence!! **Turned up brightness in ImageJ and only signal is from beads [[File:MAX_Dish3_10%25PA_4%25PFA.jpg|450px]] =Order PKP2 Padlock Probe from IDT= *To run these tests without wasting time/$/effort on padlock probe production from CA12k_Nov2014 V4 *Order single padlock probe from IDT PKP2 PKP2_3 chr12:32802524-32802525 GAGATGGCTGTCTTTTTCACACTTGG 63 GTCACCAACATGCAGCATCTTTC 63 2.719207 W GAGATGGCTGTCTTTTTCACACTTGGGTCACCAACATGCAGCATCTTTC B02203 *PKP2_controlPP: /5Phos/CCAAGTGTGAAAAAGACAGCCATCTCCTTCAGCTTCCCGATATCCGACGGTAGTGTCGAAAGATGCTGCATGTTGGTGAC
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