Hosuk:LabNotes/2013-10-23: Difference between revisions
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*epi FL images - | *epi FL images | ||
[[File:96well_2013-10-23_result_epiFL.png| | *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]] | |||
**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]] | |||
**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]] | |||
**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]] | |||
[[File:96well_2013-10-23_result_epiFL.png|900px]] | |||
Revision as of 18:10, 24 October 2013
Rolony in 96 well plate
- Compare 2 step fixing, and 1 step fixing
- Compare Anaspec and Invitrogen's a-dUTP
- Detail for beginning
- 96 Well plate
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
File:96well 2013-10-23 result epiFL.png
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.