Matt:LabNotes/2016-4-23

From ZhangLabWiki
Jump to navigation Jump to search

Test Diffusion of PA Gel Formulations[edit]

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]

  1. Prepare hydrogel mix
    1. Positive Control: Standard hydrogel + 4ul of PKP2 beads
    2. 5% PA Gel: Standard hydrogel
    3. 5% PA Gel + 4% PFA: Standard hydrogel + 4% PFA
  2. Attach 3 ~120um thick adhesives to 3 Vectabond + Bind-silane treated coverslips
  3. Add 6ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
    • Pipette away extra H2O leaving only dried beads
  4. Inject ~50ul of 200ul hydrogel mix into each dish (underneath another coverslip)
  5. Vacuum seal bag and then pump with Argon gas
  6. Let set at RT for 30min
  7. Wash with nf-H2O twice
    • 5% PA Gel + 4% PFA failed to polymerize
  • Gels are sitting in 1X PBS at 4C
  1. 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
  1. Add mix to sample and incubate for 30min at 37C
  2. Move sample to 60C and oven slowly decreases to 55C and held for ~24hr

Day 2[edit]

  1. Wash with 1X PBS once
  2. Add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C
    • Preheated to 70C before adding
  3. Wash with 2X SSC once, 1X SSC once, and 1X PBS once
  4. 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]

  1. Wash with 1X PBS twice
  2. Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) for 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris ph8.0 for 30min at RT

Imaging[edit]

Hybridize FISGA_Adpt Cy3[edit]

  1. Preheat 400ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C
  2. Add 200ul to each dish and let sit for 10min at RT
  3. Wash twice with 2X SSC

Stripped[edit]

  1. Preheat 80% formamide to 75C and then add for 15min
  2. 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