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===Procedure=== *Using Mattek dish #2 from [[Hosuk:LabNotes/2014-3-3]] that was fixed in 10% formalin ====Padlock Probe Capture==== #Strip off dye-probes Hosuk used with 80% formamide pre-heated to 75C and incubated @45C for 15min #Wash with 1X PBS twice #Anneal Padlock Probes @45C for 28hrs (Put in at 11:30a) #*1X Ampligase Buffer #*~12.6nM of each padlock probe #*0.5 U/ul Ampligase #*Prepared everything on ice and then pre-heated to 75C for 5 min before adding {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||20 |- | 30.3 pmole Agi26k0gap||58 |- | 5U/ul Ampligase||20 |- | H2O||102 |- | Total||200 |} ====Secondary RCA==== #Aspirate but don't wash #'''Pre-annealing the FISSEQ_ppRCA primer''' for the rolling circle amplification reaction. ##1ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 199ul of '''2xSSC/30% formamide''' ##Preheat primer at 60C (Tm = 55C when '''not''' in 30% formamide) ##Add 200ul and incubate at 45C for 15min. ##Aspirate, and wash using 2x SSC twice ##Aspirate, and wash using 0.1x SSC twice #Prepare the rolling circle amplification reaction mixtures on ice. ##171 ul ddH2O, ##20 ul 10x Phi29 buffer, ##2 ul 25 mM dNTP, ##4 ul 2 mM aminoallyl dUTP ##3 ul Phi29 DNA polymerase (Epicentre, low concentration) ##*200ul Total Volume #Add and incubate 20hrs at 30C. (Put in at 4:15pm 3/11/14) #Wash 1X PBS once #Add 200ul BS(PEG)9 mix to each well and incubate @RT for 1hr #Wash 1X PBS twice #Add 200ul 1M Tris pH 8.0 @RT for 30min #Wash 1X PBS twice
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