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 and dcProbes===
===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

  • 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