Matt:LabNotes/2014-9-8

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in situ Padlock Probe Capture Temp: 45C vs 60C[edit]

Procedure[edit]

  • PGP1F Samples with 1st Rolonies prepared by Hosuk
    • Fixed 9/6/14 and 1st Rolonies on 9/8/14
  • Add 100ul Padlock Probe Mix
Components Volume
5 U/ul Ampligase 10
10X Ampligase Buffer 10
100uM ppCOL1A1 2
H2O 78
Total 100
  • Incubate at 37C for 30min
  • Incubate at 45C or 60C for 26hrs
  • Wash with 1X PBS twice
  • Add 200ul 0.5uM FISSEQ_ppRCA in 2X SSC + 30% formamide (pre-heated to 75C) and incubate at 45C for 30min
  • Wash with 2X SSC twice
  • Add 200ul RCA Mix and incubate at 30C for 16hrs
    • H2O : 172uL
    • 10x Buffer : 20uL
    • dNTP : 2uL
    • a-dUTP : 4uL
    • Phi29 (100 U/µl) : 2uL
  • Wash with 1X PBS once
  • Add 200ul BS(PEG)9 (4ul BS(PEG)9 + 196ul 1X PBS) at RT for 1hr
  • Wash with 1X PBS twice
  • Add 200ul 1M Tris pH 8.0 at RT for 30min
  • Wash with 1X PBS twice and store in PBS

Imaging[edit]

Dish #1 (45C)

20X Objective Laser 4% Gain 680 Offset -1.05% Pinhole 1.00 Zoom 1.00 4096x4096 600Hz

Pos1: (67.8,41.12) z: 1552.95-1572.66 Pos2: (67.31,42.03)z: 1559.43-1578.20 Pos3: (65.91,40.54)z: 1565.21-1585.86

Dish #2 (60C)

Pos1: (71.5,40.12) z: 1361.76-1380.53 Pos2: (71.02,45.03) z: 1313.81-1334.46

Result[edit]

  • Using PISA to count features
' 45C 45C 60C 60C
1st Rolonies COL1A1 Rolonies 1st Rolonies COL1A1 Rolonies
Position 1 3,268 1,887 (58%) 1,751 1,973 (113%)
Position 2 4,341 2,259 (52%) 2,342 1,820 (78%)
Position 3 3,891 1,609 (41%)

Example images[edit]

File:20140911 45C Pos2 1stRolonies.PNG File:20140911 45C Pos2 COL1A1Rolonies.PNG

Conclusion[edit]

  • 60C may be more effective for Ampligase step but hard to compare because there were a different number of starting primary rolonies
    • Since 60C is definitely not worse we should continue with 60C padlock probe capture until a problem occurs
  • ppCOL1A1 is highly localized to nucleus, even though primary rolonies are evenly distributed in cell