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===Result for 1st try=== ====result==== *1st Padlock probes for ACTB were not seen well, too sparse and too few *2nd Padlock probes for RAB7A were many, more than ACTB. *Currently we don't know why there are not many signal on 2nd rolonies, becuase of... **Ampligase reaction were not good? or low efficiency? becuase of temperature in oven? incubation time? **Not enough padlock probes? or poor hybrizing efficiency? *need to figure out *We need to check all of 2nd rolonies, so we are going to use FITC-labeled probes which is the sequence for the region of 2nd RCA probes (488nm dye labeled CTTCAGCTTCCCGATATC) *We didn't do quantitative analysis yet, and will image this sample (RAB7A) with Confocal. ====Number of Rolonies==== *Use Matlab code (D:\My Documents\..\04. Matlab\Rolony_Counting_3.m) *Use epi FL ficture (2013-09-11) *'''ACTB : 1st Rolony --> 3931, 2nd(ACTB) --> 67''' **'''ACTB/1st = 1.70%''' *'''RAB7A : 1st Rolony --> 4085, 2nd(RAB7A) --> 204''' **'''RAB7A/1st = 4.99%''' ====Figures - epi FL==== *Rolony sample made at [[Hosuk:LabNotes/2013-6-25|2013-06-23, 3uL phi29]], 20x obj =====ACTB on 2nd rolonies===== {| {{table}} | 1st Rolonies, Cy5 ([[Media:S062319_1stRolony-Cy5_GeneACTB-Cy3_P02_Fig01_FLCy5.tif|Raw]]) || | ACTB on 2nd Rolonies, Cy3 ([[Media:S062319_1stRolony-Cy5_GeneACTB-Cy3_P02_Fig02_FLCy3.tif|Raw]]) || | Mergerd |- | [[File:S062319_1stRolony-Cy5_GeneACTB-Cy3_P02_Fig01_FLCy5_red.jpg|400px]] || | [[File:S062319_1stRolony-Cy5_GeneACTB-Cy3_P02_Fig02_FLCy3_cyan.jpg|400px]] || | [[File:Composite_ACTB_P02.jpg|400px]] | |} =====RAB7A on 2nd rolonies===== {| {{table}} | 1st Rolonies, Cy5 ([[Media:S062319_1stRolony-Cy5_GeneRAB7A-Cy3_P02_Fig02_FLCy5_EM20.tif|Raw]]) || | RAB7A on 2nd Rolonies, Cy3 ([[Media:S062319_1stRolony-Cy5_GeneRAB7A-Cy3_P02_Fig01_FLCy3_EM40.tif|Raw]]) || | Mergerd |- | [[File:S062319_1stRolony-Cy5_GeneRAB7A-Cy3_P02_Fig02_FLCy5_EM20_red.jpg|400px]] || | [[File:S062319_1stRolony-Cy5_GeneRAB7A-Cy3_P02_Fig01_FLCy3_EM40_cyan.jpg|400px]] || | [[File:Composite_RAB7A_P02.jpg|400px]] | |} ====Figures - Confocal==== *Image RAB7A sample only *20x obj, oil *Red : Cy5 (1st Rolony) *Green : Cy3 (2nd, RAB7A) *2048 x 2048 resolution, FOV : 580um x 580um with zoom 1x *Z stack(10um), and maximum projection {| {{table}} | location 1, Zoom = 1x ([[Media:Experiment.lif_Series017Snapshot1_2.tif|Raw]], [[Media:Experiment.lif_Series017Snapshot1_2_cropped|Cropped]]) || | location 2, Zoom = 2x ([[Media:Experiment.lif_Series031Snapshot All1.tif|Raw]]) |- | [[File:Confocal_RAB7A_Red-1stRolony_Green-2ndRolony_pos01_zoom1x_2.png|600px]] || | [[File:Confocal_RAB7A_Red-1stRolony_Green-2ndRolony_pos02_zoom4x.png|485px]] | |} ====='''Counting RAB7A ''' (added at 2013-10-25)===== *Rolony_Counting_10.m *1st Rolony : 29641 *2nd Rolony (RAB7A) : 2811 *Overlapped RAB7A : 791 ** 791/29641 = 2.67% **[[Media:RAB7A_Overlapped_1stRolony_2013-09-12.tif|Raw]] [[File:RAB7A_Overlapped_1stRolony_2013-09-12.png|600px]]
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