Matt:LabNotes/2014-1-28
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Checking non-specific binding of Shifted FISSEQ_2ndRCAprimerDye
- Using primary rolonies generated with RT RAB7A primers: Matt:LabNotes/2014-1-24
- 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