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]

  1. Prepare hydrogel mix
    1. Positive Control: Standard hydrogel + 2ul of PKP2 beads
    2. 10% PA Gel: Standard hydrogel
    3. 10% PA Gel + 4% PFA: Standard hydrogel + 4% PFA
  2. Attach 3 Vectabond + Bind-silane treated coverslips to 3 culture dishes
  3. Add 3ul PKP2 beads diluted in 50ul H2O to coverslips right on top of 2 magnets
  4. Inject ~70ul of 200ul hydrogel mix into each dish (underneath the plastic insert)
  5. Vacuum seal bag and then pump with Argon gas
  6. Let set at RT for 30min
  7. Wash with nf-H2O twice
  8. 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
  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]

  • 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
  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]

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]

  1. Preheat 600ul 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
  • 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]

  1. Preheat 80% formamide 2X SSC to 75C
  2. Add 200ul to Dish1 (PosCtrl) and let sit for 15min at RT
  3. Wash twice with 1X PBS
  4. 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

File:Dish2 10%PA z07 ch01.jpg

  • 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%PA.jpg

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