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===Day 4=== #Wash with 1X PBS once *LEAK! no idea why it started leaking when it has been fine until now... *Can't do RCA, just put in 4C storage <!-- #To DARTFISH slide add 1uM FISSEQ_ppRCA (1ul 200uM FISSEQ_ppRCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 55C #*Preheated to 70C before adding #Wash with 2X SSC once, 1X SSC once, and 1X PBS once #*Washed with 1X PBS another time and let sit in PBS for ~10min at RT because the RCA mix partially froze in the chilled tube holder so I had to remake the RCA mix #Add RCA mix and incubate 10.5hr at 30C {| {{table}} | align="center" style="background:#f0f0f0;"|'''Component''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||174 |- | 10X Phi29 Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | Phi 29 (low conc)||2 |- | Total||200 |} #Wash with 1X PBS once #Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate at RT for 1hr #Wash with 1X PBS twice #Add 1M Tris pH 8.0 and incubate at RT for 30min #Wash with 1X PBS twice ===Check Rolony=== #Add 0.5uM FISGA_Adpt in 2X SSC preheated to 75C #Incubate for 10min at RT #Wash with 2X SSC twice #Image (saved in 1-26-2015) #Strip with 80% formamide preheated to 75C #Wash with 1X PBS twice -->
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