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Matt:LabNotes/2014-4-30
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==MALAT1 Rolonies and EDTA== *Test to see if rolonies quenched with TE (Tris + EDTA) are easier for padlock probes to hybridize to **Use artificial MALAT1 rolonies *Quantify the amplification of Secondary Rolonization from Primary Rolonies ===Procedure=== #Add 140ul 100nM MALAT1_template to each well #*Pre-heat to 85C #*Incubate @RT for 15 min #Aspirate & Wash once with 1X PBS #Capture with 10pM ppMALAT1 #*Incubate @45C for 20 hours {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||60 |- | 1nM ppMALAT1||6 |- | 5 U/ul Ampligase||60 |- | H2O||474 |- | Total||600 |} #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 #*To A3-A5: 1M Tris pH 8.0 #*To D3-D5: 0.9M Tris + 50mM EDTA ph 8.0 (45mL 1M Tris + 5mL 500mM EDTA) #Wash 1X PBS twice #Add 1uM dcProbe2-Cy3 in 30% formamide + 2X SSC #*Preheat to 75C for 5min #*Incubate @RT for 10min #Wash 2X SSC twice #Image #Strip w/ 80% formamide in 2X SSC #*Pre-heat to 75C #*Incubate @RT for 15min #Wash with ddH2O twice ===Results=== *PISA wasn't able to count many images due to not reaching minimum number of features **Used bwlabel to count features after applying a threshold of 9,000 to make a bw image {| {{table}} | align="center" style="background:#f0f0f0;"|'''Well''' | align="center" style="background:#f0f0f0;"|'''Position''' | align="center" style="background:#f0f0f0;"|'''Rolonies counted by PISA''' | align="center" style="background:#f0f0f0;"|'''Rolonies counted by bwlabel (threshold 9000)''' |- | A3||1||N/A||55 |- | A3||2||140||141 |- | A4||3||N/A||69 |- | A4||4||N/A||104 |- | A5||5||N/A||66 |- | A5||6||N/A||127 |- | D3||7||N/A||41 |- | D3||8||N/A||51 |- | D4||9||N/A||48 |- | D4||10||137||146 |- | D5||11||139||140 |- | D5||12||128||127 |} *Continued on [[Matt:LabNotes/2014-5-7]]
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