Hosuk:LabNotes/2013-10-23
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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
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.