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===Procedure=== ====Anneal Padlock Probes==== *1X Ampligase Buffer *2uM of each padlock probe *0.5 U/ul Ampligase *Incubate @ 45C for 24 hours **Wrapped in parafilm and put in ziploc with wet towel =====Fixed Template - 25U Ampligase (C9 & F9)===== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 200uM ppMALAT1||1 |- | 5U/ul Ampligase||10 |- | H2O||79 |- | Total||100 |} =====Fixed Template - 50U Ampligase (C10 & F10)===== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 200uM ppMALAT1||1 |- | 5U/ul Ampligase||20 |- | H2O||69 |- | Total||100 |} =====1st Rolony (E6 & F6)===== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | 10X Ampligase Buffer||10 |- | 200uM ppMALAT1||1 |- | 200uM ppACTB||1 |- | 200uM ppRAB7A||1 |- | 5U/ul Ampligase||10 |- | H2O||77 |- | Total||100 |} =====Empty Negative Control (D7 & E7)===== *Not going to waste Ampligase since it's highly unlikely anything will be ligated, but will do RCA in these wells =====Pre-circularized Padlock Probes (C7 & C8)===== *Combined all the pre-circle ppMALAT1 made on: [[Matt:LabNotes/2014-2-10]] into 64ul *75% EtOH for 10 minutes *Wash with 1X PBS 2 times *0.1N HCl for 2 minutes *Wash with 1X PBS 3 times *Add 32ul to C7 & C8 =====Fixed Pre-circularized Padlock Probes (F7 & F8)===== *Not going to do anything during Ampligase step ====Secondary RCA==== #Aspirate each well but don't wash #*F7 well was dried up #'''Pre-annealing the FISSEQ_ppRCA primer''' for the rolling circle amplification reaction. ##3ul of RCA primer(200 uM, GATATCGGGAAGCTGA*A*G) in 597ul of '''2xSSC/30% formamide''' ##Preheat primer at 60C (Tm = 55C when '''not''' in 30% formamide) ##Add 50ul to each well, 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. ##513 ul ddH2O, ##60 ul 10x Phi29 buffer, ##6 ul 25 mM dNTP, ##12 ul 2 mM aminoallyl dUTP ##9 ul Phi29 DNA polymerase (Epicentre, low concentration) ##*600ul Total Volume #Add 50ul to each well and incubate overnight at 30C. (Put in at 7pm 2/26/14) #Wash 1X PBS once #Add 50ul BS(PEG)9 mix to each well and incubate @RT for 1hr (12ul BS(PEG)9 + 588ul 1X PBS) #Wash 1X PBS twice #Add 50ul 1M Tris pH 8.0 @RT for 1hr #Wash 1X PBS twice ====Detection==== *To all wells add 50ul 1uM dcProbe2-Cy3 (6ul 100uM dcProbe2-Cy3 + 180ul 100% formamide + 60ul 20X SSC + 354ul H2O preheated to 75C) **Strip E6 & F6 with 80% formamide @ 37C *To E6 & F6 add 50ul 1uM dcProbe0-Cy3 (10ul 10uM dcProbe0-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C) **Strip with 80% formamide @37C *To E6 & F6 add 50ul 1uM dcProbe1-Cy3 (10ul 10uM dcProbe1-Cy3 + 30ul 100% formamide + 10ul 20X SSC + 50ul H2O preheated to 75C) *'''Images saved under 2-27-2014 folder'''
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