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*[[Hosuk:Lab_Notes|LabNote]] ===Test : +/- a-dUTP, and long RT incubation at 4C=== *Continued from [[Hosuk:LabNotes/2014-5-6|05/06]] ====Procedure==== *05/06 *#10% Formalin for 15min at RT(25C) --> done at CERC *#Wash with PBS 3 times --> done at CERC *#Move samples to the lab at PFBH *#Add 0.25% Triton X-100 in DEPC-PBS and incubate for 10min at room temp. *#mean while prepare RT mix and put the mix on an ice *#Aspirate 0.25% Triton X-100 in DEPC-PBS and wash with PBS 3 times *#Add RT mix and put in 4C, incubate for 15min. (1hr. for other 2 sample of target specific primers) *#Move samples to 37C and incubate for 12hr. *05/07 *#Wash with PBS once *#Prepare 18uL of BS(PEG)9 in 782uL PBS (for 4 dishes, volume per each dish is 200uL) *#add 200uL BS(PEG)9 mix, incubate for 1hr at RT *#Wash with PBS once, add Tris pH 8.0, incubate for 30min *#Add RNase H mix, incubate for 1hr. at 37C *#*RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O *#Wash with nucleaus-free H2O twice *#Add CircLigase II mix, incubate at 60C for 3hr. *#*H2O 128uL *#*Buffer 20uL *#*50mM MnCl2 10uL *#*5M Betaine 40uL *#*CircLigaseII 2uL *#RCA Primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr. *#* 1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide *#Aspirate and add 2XSSC, incubate at 60C for 10min *#Aspirate and Wash with 2X SSC, 1X SSC and PBS once each *#Prepare RCA reaction mix *#*H2O 168uL *#*Buffer 20uL *#*25mM dNTP 4uL *#*2mM a-dUTP 4uL *#*Phi29 4uL *#Add RCA mix, incubate at 30C for 20hr *05/08 *#Wash with PBS once *#Prepare 18uL of BS(PEG)9 in 782uL PBS (for 4 dishes, volume per each dish is 200uL) *#add 200uL BS(PEG)9 mix, incubate for 1hr at RT *#Wash with PBS once, add Tris pH 8.0, incubate for 30min *#Wash with PBS once *#Prepare detection Probe : Final Conc. 2.5uM --> 5uL of 100uM Cy3-Adapt in 200uL (2xSSC + 30% Formamide) *#Preheat detection Probe at 80C for 5min *#Add to sample and incubate at RT for 10min *#Wash two times for 1 minute each with 1 mL 1X PBS *#Imaging
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