Hosuk:LabNotes/2013-10-23: Difference between revisions

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*epi FL images
*epi FL images
*Raw images  
*Raw images  
**C5 : [[Media:96well_S1-C5_pos1_fig01_Cy5_EM40_exp0.1.tif|Cy5]], [[Media:96well_S1-C5_pos1_fig01_FITC_EM40_exp0.1.tif|ATTO488]]
**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_fig01_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-C5_pos1_fig01_Cy5_EM40_exp0.1.tif|Cy5]], [[Media:96well_S9-C5_pos1_fig01_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-C5_pos1_fig01_Cy5_EM40_exp0.1.tif|Cy5]], [[Media:96well_S13-C5_pos1_fig01_FITC_EM40_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]]





Latest revision as of 18:14, 24 October 2013

Rolony in 96 well plate[edit]

  • Compare 2 step fixing, and 1 step fixing
  • Compare Anaspec and Invitrogen's a-dUTP
  • Detail for beginning
  • 96 Well plate

File:96well 2013-10-23.png


Result[edit]
  • 1 step fixing samples were good, 2 step fixing samples had less rolonies.
  • There were no differences between Anaspec and Invitrogen's a-dUTP



File:96well 2013-10-23 result epiFL.png


Issues of Gene detection with Padlock probe[edit]

  • 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.