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====Continued protocol with DARTFISH2==== *Only had enough padlock probes for 1 dish so chose DARTFISH2 7.2.2015 #Add RNase Mix (168ul H2O + 20ul RNase H Buffer + 2ul Riboshredder + 10ul RNase H) and incubate 1hr at 37C #Wash with 2ml H2O twice #Prepare Ampligase mixture '''on ice''' #*Suppressor Oligos 100nM (labeled V4 supp v2 100nm each ALL (40+5) 7.6.15) - 25ul #**10ul V4 5supp 10uM each + 100ul V4 40supp 1uM each + 890ul H2O = 1000ul 100nM each #*Ampligase Buffer 10X - 10ul #*[[Matt:LabNotes/2015-6-29#Qubit_Quantification | Padlock Probes 227nM]] - 38ul (should be 44ul for 100nM total) #**Heat to 90C first and snap cool on ice block (FORGOT TO HEAT) #*H2O - 17ul #*Ampligase 5U/ul - 10ul #Add Ampligase mix to sample and incubate 3hr at 60C then 21hr at 55C (incubator takes a long time to cool from 60C to 55C with door closed) #*Middle of glass was dry? #Wash with 1X PBS once #Add 200ul (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C #Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS #Prepare RCA reaction mix '''on ice''' #*H2O - 174ul #*Phi29 Buffer 10X - 20ul #*dNTP 25mM - 2ul #*aa-dUTP 4mM - 2ul #*Phi29 DNA polymerase 100U/ul - 2ul #Add RCA mix and incubate at 30C overnight (~18hrs) #Wash with 1X PBS once #Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS prepared on ice) to sample and incubate 1hr at RT #Wash with 1X PBS twice #Add 200ul 1M Tris (pH 8.0) for 30min at RT #Wash with 1X PBS twice and store at 4C<!-- #Add 0.5uM FISGA_Adpt (1ul 100uM FISGA_Adpt + 199ul 2XSSC + 30% formamide) pre-heated to 75C and incubate 10min at RT #Wash with 2X SSC twice and add 1ml 2X SSC for imaging #Strip with 80% formamide pre-heated to 75C and incubate 15min at RT #Wash with 1X PBS twice and store in 2ml PBS-->
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