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===DARTFISH=== *Sample: RT 15min **Labeled "DARTFISH1 6.8.2015" *Sample: 37C 10min **Labeled "DARTFISH2 6.8.2015" #Wash the cells with 1ml of PBS (RNase free) 3 times #Prepare RT mixture '''on ice''' #*DEPC-H2O - 318ul #*M-MuLV RT Buffer 10X - 40ul #*dNTP 25mM - 4ul #*aa-dUTP 4mM - 4ul #*RT primer 100uM - 10ul #**Nonamer #*RNase inhibitor 40U/ul - 4ul #*M-MuLV reverse transcriptase 100U/ul - 20ul #Add RT mixture and incubate for 10min at 4C #Transfer sample to 37C overnight (~15hrs) #Wash with 1X PBS once #Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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 #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 (2X volume) '''on ice''' #*H2O - 110ul #*Ampligase Buffer 10X - 20ul #*[[Matt:LabNotes/2015-5-11#Qubit_Quantification | Padlock Probes 461nM]] - 50ul #**Heat to 90C first and snap cool on ice block #*Ampligase 5U/ul - 20ul #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) #*3:20p-3p #Wash with 1X PBS once #Add 200ul (1ul 100uM 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 (2X volume) '''on ice''' #*H2O - 348ul #*Phi29 Buffer 10X - 40ul #*dNTP 25mM - 4ul #*aa-dUTP 4mM - 4ul #*Phi29 DNA polymerase 100U/ul - 4ul #Add RCA mix and incubate at 30C overnight (~14hrs) #Wash with 1X PBS once #Add 300ul cold BS(PEG)9 (6ul stock BS(PEG)9 + 294ul 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 #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 ====Rolony Check 6/11/2015==== *Much more rolonies than FISSEQ *DARTFISH1 [[File:20150611_MotorNeur_DARTFISH1_Pos1_FISGAadpt_MIP_overlay_BF.jpg|400px]] *DARTFISH2 [[File:20150611_MotorNeur_DARTFISH2_Pos1_FISGAadpt_MIP_overlay_BF.jpg|400px]] ====Decoding Results 6/17/2015==== *[[Matt:LabNotes/2015-6-17]]
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