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*[[Hosuk:Lab_Notes|LabNote]] ===Rolony in 96 well plate=== *Compare 2 step fixing, and 1 step fixing *Compare Anaspec and Invitrogen's a-dUTP *[[Hosuk:LabNotes/2013-10-21|Detail for beginning]] *96 Well plate [[File:96well_2013-10-23.png|500px]] =====Result===== *1 step fixing samples were good, 2 step fixing samples had less rolonies. *There were no differences between Anaspec and Invitrogen's a-dUTP *epi FL images *Raw images **C5 : [[Media:96well_S1-C5_pos1_fig01_Cy5_EM40_exp0.1.tif|Cy5]], [[Media:96well_S1-C5_pos1_fig02_FITC_EM40_exp0.1.tif|ATTO488]] **D5 : [[Media:96well_S5-D5_pos1_fig01_Cy5_EM40_exp0.1.tif|Cy5]], [[Media:96well_S5-D5_pos1_fig02_FITC_EM40_exp0.1.tif|ATTO488]] **E5 : [[Media:96well_S9-E5_pos1_fig01_Cy5_EM15_exp0.1.tif|Cy5]], [[Media:96well_S9-E5_pos1_fig02_FITC_EM15_exp0.1.tif|ATTO488]] **F5 : [[Media:96well_S13-F5_pos1_fig01_Cy5_EM15_exp0.1.tif|Cy5]], [[Media:96well_S13-F5_pos1_fig02_FITC_EM15_exp0.1.tif|ATTO488]] [[File:96well_2013-10-23_result_epiFL.png|900px]] ===Issues of Gene detection with Padlock probe=== *The gene-specific sequence for detection could not be found in 1st rolonies because. *Random hexamers as RT probe anneal randomly in mRNA (as well as any RNAs) so the generated cDNA may or may not have the detection sequence. *And RTase can't proceed when it meet primer while synthesis, thus the detection sequence cannot be included if any primer anneals in anywhere of detection sequence region. *Since our padlock probe use about 40 base sequence and mRNA of ACTB is about 1900, so the chance that the cDNA has the detection sequence lower than 1% (considering the reason mentioned upper sentence) *Ask the raw sequencing data from Church group and figure out how many ACTB rolonies out of all rolonies, how many rolonies have the detection sequence, etc... *Use multiple number of specific sequence for targeting ACTB, in order to anneal the location close to the sequence *And try to make "semi-random" 7-, 8- or 9- primer to have high enrichment from the location where cDNA could have the detection sequence.
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