Matt:LabNotes/2014-1-28: Difference between revisions
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>Mzcai (Created page with "===Checking non-specific binding of Shifted FISSEQ and dcProbes===") |
>Mzcai |
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===Checking non-specific binding of Shifted FISSEQ | ===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==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''RT-RAB7A''' | |||
| align="center" style="background:#f0f0f0;"|'''Primary Rolony Cy3''' | |||
| align="center" style="background:#f0f0f0;"|'''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 |
Revision as of 01:50, 1 February 2014
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