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*[[Hosuk:Lab_Notes|LabNote]] ===Human Brain Tissue : CA12k_Nov2014 V4 & V7 : 1st Try=== ====3rd day==== *Continued from [[Hosuk:LabNotes/2015-2-4|02/04]] *02/05 *#For V4 & V7 samples: RCA Primer, final concentration : 0.5uM in 200uL (2x SSC) at 60C for 1hr. *#1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide *#Wash with 2XSSC twice, and 1X SSC twice *#Prepare RCA reaction mix *#*H2O 172uL *#*Buffer 20uL *#*25mM dNTP 2uL *#*2mM a-dUTP 4uL *#*Phi29(low conc.) 2uL *#Add RCA mix, incubate at 30C overnight *#For 1st Rolony generation : Wash with PBS once *#Prepare 4uL of BS(PEG)9 in 196uL PBS *#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. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (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
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