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===Dye labeled Probe Annealing Test β with dcProbe3-Cy3 : Non-specific binding=== *Figure out lower limit by Formamide concentration : to minimize non-specific binding *dcProbe3-Cy3 does not have target in rolony, so we can see how many spots due to the non-specific binding. *Done @ 11/19 ====Procedure==== *20x obj, 2048 x 2048 resolution, 16bit, *Scan Freq. 600Hz --> Image quality is not different from 400Hz. *Sample : MatTek Dish, made at 07/09 *Each stripping, and annealing : 15min incubation @ RT *Each time the concentration of the dye-labeled probe is 1uM, *The volume added is 100uL. *Process Steps Process Step Step 0 : Strip Step 1 : Cy3 dcProbe3 without Formamide+ 2x SSC ο 160 Step 2 : Strip ο ~34 Step 3 : Cy3 dcProbe3 in 30% Formamide + 2x SSC ο 67 Step 4 : Strip Step 5 : Cy3 dcProbe3 in 40% Formamide + 2x SSC ο 66 *#Step 0 : Strip *#Step 1 : Cy3 dcProbe3 without Formamide+ 2x SSC *#Step 2 : Strip *#Step 3 : Cy3 dcProbe3 in 30% Formamide + 2x SSC *#Step 4 : Strip *#Step 5 : Cy3 dcProbe3 in 40% Formamide + 2x SSC ====Result==== *30% seemed optimal concentration for less non-specific binding as well as for target-specific binding.. *Need to get 3 data point to confirm those results (for the future when I set up an automation system. =====Spot Count===== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Formamide (%)''' | align="center" style="background:#f0f0f0;"|'''Spot Count''' |- | align="center" | Strip || align="center" | 34 |- | align="center" | 0 || align="center" | 160 |- | align="center" | 30 || align="center" | 67 |- | align="center" | 40 || align="center" | 66 |} [[File:Chart_DyeProbeTest_dcProbe3_Step1-2-3-5.png|800px]] *'''Raw images''' **[[Media:DyeProbeTest.lif_MatTek20130709_Step1_dcProbe3_Cy3_ch00_MIP.tif|Step1 (Cy3, ]][[Media:DyeProbeTest.lif_MatTek20130709_Step1_dcProbe3_Cy3_ch01_MIP.tif|BF)]], [[Media:DyeProbeTest.lif_MatTek20130709_Step2_Strip_Cy3_ch00_MIP.tif|Step2]], [[Media:DyeProbeTest.lif_MatTek20130709_Step3_dcProbe3_Formamide30_Cy3_ch00_MIP.tif|Step3]], [[Media:DyeProbeTest.lif_MatTek20130709_Step4_Strip_Cy3_ch00_MIP.tif|Step4]], [[Media:DyeProbeTest.lif_MatTek20130709_Step5_dcProbe3_Formamide40_Cy3_ch00_MIP.tif|Step5]]
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