Matt:LabNotes/2016-4-23
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Test Diffusion of PA Gel Formulations[edit]
- Using PKP2 Beads as targets
- PKP2_controlPP
Dilute PKP2_controlPP[edit]
- Resuspended to 10uM with 450ul H2O
- Serial dilute to 10nM
- 10:1 dilution 3 times
Experiment Plan[edit]
- 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[edit]
Day 1[edit]
- Prepare hydrogel mix
- Positive Control: Standard hydrogel + 4ul of PKP2 beads
- 5% PA Gel: Standard hydrogel
- 5% PA Gel + 4% PFA: 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
Component | 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[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]
- 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]
Hybridize FISGA_Adpt Cy3[edit]
- 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[edit]
- Preheat 80% formamide to 75C and then add for 15min
- Wash twice with 1X PBS
Align Images and Subtract Stripped[edit]
- Dish 1: PosCtrl
File:Cropped MAX PosCtrl BGsubtracted.jpg
- Dish 2: No Beads Area
File:Cropped MAX 5% NoBeads BGsubtracted.jpg
- Dish 2: Beads
File:Cropped MAX 5% Beads BGsubtracted.jpg
Conclusion[edit]
- 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