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==Imaging== *Used Gain and Laser power usually used for DARTFISH decoding **ch00: Cy5 **ch01: Cy3 **ch02: BF *Pos1: NegCtrl with no beads **No signal in any images, GOOD! *Pos2: Scattered beads *Pos3: Scattered beads ====Before Hybridization==== *No fluorescent signal anywhere *Pos 2: [[File:Pos2_Before_z05_ch02.jpg|450px]] *Pos 3: [[File:Pos3_Before_z06_ch02.jpg|450px]] ====Hybridize FISGA_Adpt Cy3==== #Preheat 200ul 0.5uM FISGA_Adpt_Cy3 in 30% formamide 2X SSC to 75C #Add 200ul to each dish and let sit for 10min at RT #Wash twice with 2X SSC {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | align="right" width="51" height="14" | | width="51" | Cy3 | width="51" | Cy5 |- style="font-size:11pt" valign="bottom" | height="14" | Pos 2 | [[File:MAX_Pos2_FISGA_ch01.jpg|450px]] | [[File:MAX_Pos2_FISGA_ch00.jpg|450px]] |- style="font-size:11pt" valign="bottom" | height="14" | Pos 3 | [[File:MAX_Pos3_FISGA_ch01.jpg|450px]] | [[File:MAX_Pos3_FISGA_ch00.jpg|450px]] |} ====Strip==== #Preheat 80% formamide 2X SSC to 75C #Add 200ul to Dish1 (PosCtrl) and let sit for 15min at RT #Wash twice with 1X PBS #Add 500ul 2x SSC to image *Pos 2: [[File:MAX_Pos2_Stripped_ch01.jpg|450px]] *Pos 3: [[File:MAX_Pos3_Stripped_ch01.jpg|450px]] *Stripping of dye is incomplete **Need to wash more, strip longer, or make gel thinner so diffusion is faster *Some locations are stripped better than others ====Hybridize Cycle5 Cy3 & Cy5==== #Preheat 200ul 0.5uM Cycle5_Cy3 and 0.5uM Cycle5_Cy5 in 30% formamide 2X SSC to 75C #*Expect Cy5 signal only, no Cy3 #Add 200ul to each dish and let sit for 10min at RT #Wash twice with 2X SSC {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="font-size:11pt" valign="bottom" | align="right" width="51" height="14" | | width="51" | Cy3 | width="51" | Cy5 |- style="font-size:11pt" valign="bottom" | height="14" | Pos 2 | [[File:MAX_Pos2_Cycle5_ch01.jpg|450px]] | [[File:MAX_Pos2_Cycle5_ch00.jpg|450px]] |- style="font-size:11pt" valign="bottom" | height="14" | Pos 3 | [[File:MAX_Pos3_Cycle5_ch01.jpg|450px]] | [[File:MAX_Pos3_Cycle5_ch00.jpg|450px]] |} *Specific Cy5 signal that wasn't there before *While most spots are on top of beads, they are definitely rolony specific because there are some spots that aren't near beads and these coincide with spots seen with FISGA_Cy3 dye
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