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==in situ Padlock Probe Capture Temp: 45C vs 60C== ===Procedure=== *PGP1F Samples with 1st Rolonies prepared by Hosuk **Fixed 9/6/14 and 1st Rolonies on 9/8/14 *Add 100ul Padlock Probe Mix {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''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/Β΅l) : 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=== 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=== *Using PISA to count features {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''45C''' | align="center" style="background:#f0f0f0;"|'''45C''' | align="center" style="background:#f0f0f0;"|'''60C''' | align="center" style="background:#f0f0f0;"|'''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==== [[File:20140911_45C_Pos2_1stRolonies.PNG]] [[File:20140911_45C_Pos2_COL1A1Rolonies.PNG]] ===Conclusion=== *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
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