Hosuk:LabNotes/2013-10-23: Difference between revisions
Jump to navigation
Jump to search
>Hosuki78 No edit summary |
>Hosuki78 No edit summary |
||
Line 13: | Line 13: | ||
*1 step fixing samples were good, 2 step fixing samples had less rolonies. | *1 step fixing samples were good, 2 step fixing samples had less rolonies. | ||
*There were no differences between Anaspec and Invitrogen's a-dUTP | *There were no differences between Anaspec and Invitrogen's a-dUTP | ||
*epi FL images - each wells (C5, D5, E5, and F5) | |||
[[File:96well_2013-10-23_result_epiFL.png|800px]] | |||
Revision as of 18:04, 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
- epi FL images - each wells (C5, D5, E5, and F5)
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.