Matt:LabNotes/2014-1-28

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Checking non-specific binding of Shifted FISSEQ_2ndRCAprimerDye

  • Added 50ul 1uM FISSEQ_Adpt to wells in column 9 (20ul 20X SSC + 60ul 100% formamide + 118ul H2O + 2ul 100uM FISSEQ_Adpt)
  • Added 50ul 2uM Shifted FISSEQ_2ndRCAprimerDye to wells in column 10 (20ul 20X SSC + 60ul 100% formamide + 118ul H2O + 2ul 200uM Shifted FISSEQ_2ndRCAprimerDye)

Results

RT-RAB7A Primary Rolony Cy3 Shifted FISSEQ 488
220 711 342
300 357 136
510 4449 396
1000 13295 20910
  • RT-RAB7A 1000nt wells were very saturated
  • Besides RT-RAB7A 510nt, the number of rolonies is comparable

Conclusion

  • Shifted FISSEQ_2ndRCAprimerDye has significant hybridization to non-specific cell structures OR to primary rolonies
    • Most likely to primary rolonies because of the correlation in # of signals between Cy3 and 488