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==Artifical Rolonies== ===Procedure=== #Add 140ul 100nM MALAT1_template to each well #*Pre-heat to 85C #*Incubate @RT for '''75 min''' #**C4 & C5 Incubate @RT for '''60 min''' #Aspirate NO WASH #Capture with 100pM ppMALAT1 #*Incubate @45C for 24 hours (6:30p-6:30p) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||80 |- | 2nM ppMALAT1||40 |- | 5 U/ul Ampligase||80 |- | H2O||600 |- | Total||800 |} #Aspirate NO WASH #Add RCA primer: 4ul FISSEQ_ppRCA in 796 2X SSC/30% (1uM final concentration) #*Pre-heat to 60C #*Add 100ul @45C for 15min #*Wash 2X SSC twice #*Wash 0.1X SSC twice #Add RCA mix #*Incubate 21hrs @ 30C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||684 |- | 10X Phi29 Buffer||80 |- | 25mM dNTP||8 |- | 2mM aa-dUTP||16 |- | Phi29||12 |- | Total||800 |} #Wash 1X PBS once #16ul BS(PEG)9 in 784ul 1X PBS #*Incubate @RT 1hr #Wash 1X PBS twice #Quench with 1M Tris pH 8.0 @RT 1hr #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 -->
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