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===Experiment=== *Sample: BA8_S2_V4_3-4-2015 *Dye-probe: 0.5uM FISGA_Adpt (Cy3) **3 Conditions: ***Dye 1) 0.5uM in 30% formamide + 2X SSC (1ul 100uM dye + 20ul 20X SSC + 60ul formamide + 119ul H2O = 200ul) ***Dye 2) 0.5uM in 10% dextran sulfate + 30% formamide + 2X SSC (0.5ul 100uM dye + 10ul 20X SSC + 30ul formamide + 20ul 50% dextran sulfate + 39.5ul H2O = 100ul) ***Dye 3) 0.5uM in 10% dextran sulfate + 10% formamide + 2X SSC (0.5ul 100uM dye + 10ul 20X SSC + 10ul formamide + 20ul 50% dextran sulfate + 59.5ul H2O = 100ul) ====Imaging Order==== #Strip sample with 80% formamide #Add 100ul Dye 1 to find 4 positions and set laser power & gain to get minimal saturated pixels while broadest distribution of intensities #*1% laser (552nm wavelength), [V] = 590, % offset = -1.00 #Image (Named Step 1) #Strip #Add 100ul Dye 1 and Image (Named Step 2) #Strip #Add 100ul Dye 2 and Image (Named Step 3) #Strip #Add 100ul Dye 3 and Image (Named Step 4) #Strip *For all: **preheat dyes & 80% formamide in 2X SSC to 75C **incubate @RT 10min to hybridize and then wash with 2X SSC twice **incubate @RT 15min to strip and then wash with 1X PBS twice *Take 10 z-stack images for each of 4 positions (40 images for each step)
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