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==ppMALAT1_dcProbe1 Capture of MALAT1 Rolonies +/- EDTA== *Continued from [[Matt:LabNotes/2014-4-30]] *Test to see if rolonies quenched with TE (Tris + EDTA) are easier for padlock probes to hybridize to *Quantify the amplification of Secondary Rolonization from Primary Rolonies ===Procedure=== *Add 100ul ppMALAT1_dcProbe1 with 0.5 U/ul Ampligase **A3&D3: 10pM **A4&D4: 50pM **A5&D5: 2uM **Incubate @45C for 24 hours '''10pM''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||20 |- | 5 U/ul Ampligase||20 |- | 1nM ppMALAT1_dcProbe1||2 |- | H2O||158 |- | Total||200 |} '''50pM''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||20 |- | 5 U/ul Ampligase||20 |- | 1nM ppMALAT1_dcProbe1||10 |- | H2O||150 |- | Total||200 |} '''2uM''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||20 |- | 5 U/ul Ampligase||20 |- | 100uM ppMALAT1_dcProbe1||4 |- | H2O||156 |- | Total||200 |} *Aspirate NO WASH *Add RCA primer: 3ul FISSEQ_ppRCA in 597 2X SSC/30% **Pre-heat to 60C **Add 100ul @45C for 15min **Wash 2X SSC twice **Wash 0.1X SSC twice *Add RCA mix **Incubate 22hrs @ 30C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||513 |- | 10X Phi29 Buffer||60 |- | 25mM dNTP||6 |- | 2mM aa-dUTP||12 |- | Phi29||9 |- | Total||600 |} *Wash 1X PBS once *12ul BS(PEG)9 in 588ul 1X PBS **Incubate @RT 1hr *Wash 1X PBS twice *Quench with 1M Tris pH 8.0 @RT 1hr *Wash 1X PBS twice *Add 1uM dcProbe1-Cy3 in 30% formamide + 2X SSC **Preheat to 75C for 5min **Incubate @RT for 10min *Wash 2X SSC twice *Image **Used Pinhole: 2.0 ===Results=== *Saved in 5-8-2014 *Used Pinhole = 2.0 **Tried Pinhole = 1.0 for Pos9 the count of rolonies was 2,801 (80% of Pinhole 2.0) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Well''' | align="center" style="background:#f0f0f0;"|'''Position''' | align="center" style="background:#f0f0f0;"|'''Rolonies''' |- | A3||1||930 |- | A3||2||1562 |- | A4||3||3134 |- | A4||4||4352 |- | A5||5||4997 |- | A5||6||9189 |- | D3||7||1272 |- | D3||8||1062 |- | D4||9||3480 |- | D4||10||3758 |- | D5||11||3315 |- | D5||12||1909 |} [[File:MALAT1_2ndRol_Results.JPG]] ===Conclusion=== *Padlock probe capture (followed by RCA) of primary rolonies greatly amplifies signal *EDTA does not help with padlock probe capture *Increasing concentration above 50pM does not improve 2nd rolony yield *Based on [[Matt:LabNotes/2014-4-30#Results]] there's good correlation with number of primary rolonies and number of secondary rolonies
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