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====Add circular DNA (~50pmole) in fixed cells and run RCA in the oven==== *Produce circular DNA (50pmole input!) as same one made at 05/ *20uL of CircLigase II product + 30uL 2x SSC in the fixed cell dish *Run RCA in the oven (set temp: 33C) for 19 hour *After RCA, *#Add 20 ul 250 mM BS(PEG)9 in 1 ml PBS for 1 hr at RT. *#Wash with PBS once *#Block with 1M Tris pH 8.0 for 30 min. *#Wash with PBS once *#Add 5 ul Riboshredder (Epicentre)+ 5ul RNase H(Enzymatics) in 190ul RNase H buffer(Enzymatics) for 1 hr at 37C. *#Rinse the entire well with ddH2O twice to remove traces of phosphate. *#Prepare 100uM of Cy3 labeled probe (Cy3-TCTTCAGCGTTCCCTAGA) *#2uL of 100uM probes in 200uL 2x SSC *#Pre-heated probe solution between 60C and 80C for 5min *#Add to cells at room temp, and let it cool down over 5min *#Wash with 2x SSC (2mL, but 200uL work too) twice and image! ====Result==== *RCA rolony signal were from Cy3 and FITC channel very well. *There were a lot of big strong spots, which were seen every time before, and I don’t think that’s rolonies, *they looked cluster or chuck of RCA products so the Cy3 adpaters were bound there. *Anyway, FITC channel represents Rolonies well… *Let’s try Confocal imaging! *Let’s move on a real protocol, from RT to RCA! =====20x, Position 2===== *Raw images : [[Media:S4_50pmoleCircDNA_20x_pos02_fig01_Cy3.tif|Cy3]], [[Media:S4_50pmoleCircDNA_20x_pos02_fig02_FITC.tif|FITC]], [[Media:S4_50pmoleCircDNA_20x_pos02_fig03_Cy5.tif|Cy5]], [[Media:S4_50pmoleCircDNA_20x_pos02_fig04_BF.tif|BF]] [[File:S4_20x_pos2_052013.png|800px]] =====40x Oil, Position 2===== *Raw images : [[Media:S4_50pmoleCircDNA_40xOil_pos02_fig01_Cy3.tif|Cy3]], [[Media:S4_50pmoleCircDNA_40xOil_pos02_fig02_FITC.tif|FITC]], [[Media:S4_50pmoleCircDNA_40xOil_pos02_fig03_Cy5.tif|Cy5]], [[Media:S4_50pmoleCircDNA_40xOil_pos02_fig04_BF.tif|BF]] [[File:S4_40xOil_pos2_052013.png|800px]] =====40x Oil, Position 3===== *Raw images : [[Media:S4_50pmoleCircDNA_40xOil_pos03_fig01_Cy3.tif|Cy3]], [[Media:S4_50pmoleCircDNA_40xOil_pos03_fig02_FITC.tif|FITC]], [[Media:S4_50pmoleCircDNA_40xOil_pos03_fig03_Cy5.tif|Cy5]], [[Media:S4_50pmoleCircDNA_40xOil_pos03_fig04_BF.tif|BF]] [[File:S4_40xOil_pos3_052013.png|800px]]
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