Matt:LabNotes/2016-4-11
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Test Diffusion of PA Gel Formulations[edit]
- Using PKP2 Beads as targets
- CA12kNov2014 V4 Padlock Probes
Experiment Plan[edit]
- 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[edit]
Day 1[edit]
- Prepare hydrogel mix
- Positive Control: Standard hydrogel + 2ul of PKP2 beads
- 10% PA Gel: Standard hydrogel
- 10% PA Gel + 4% PFA: 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
- 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?)
Component | 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[edit]
- 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
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
Day 3[edit]
- 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[edit]
Dish 1: Positive Control[edit]
- Beads well dispersed among many z layers
- No z-slice has them all in focus
Hybridize FISGA_Adpt Cy3[edit]
- 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
- 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.jpgFile:MAX Dish1 PosCtrl FISGA Adpt Pos2.jpg
Strip[edit]
- 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
- Position 1 (Cy5)
File:MAX Dish1 PosCtrl Stripped Cy5 Pos1.jpg
Hybridize Cycle5 Cy3 & Cy5[edit]
- Only Cy5 should show signal
- Position 1 (Cy3)
File:MAX Dish1 PosCtrl Cycle5 Cy3 Pos1.jpg
- Position 1 (Cy5)
- Good there's signal when there wasn't before!
File:MAX Dish1 PosCtrl Cycle5 Cy5 Pos1.jpg
Dish 2: 10% PA[edit]
- Beads are clearly in focus in single z-plane
- High density where magnet was
- 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
Dish 3: 10% PA 4% PFA[edit]
- Beads are clearly in focus in single z-plane
- High density where magnet was
File:Dish3 10%PA 4%PFA z07 ch01.jpg
- Hybridized FISGA_Adpt_Cy3 but No Fluorescence!!
- Turned up brightness in ImageJ and only signal is from beads
File:MAX Dish3 10%PA 4%PFA.jpg
Order PKP2 Padlock Probe from IDT[edit]
- 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