Matt:LabNotes/2014-1-28: Difference between revisions

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>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
**This can be further explored by seeing of adding RT primer (which has complementary sequence to RCA primer site) can reduce hybridization of Shifted FISSEQ, which would indicated Shifted FISSEQ is hybridizing to the RCA primer sequence of primary rolonies: [[Matt:LabNotes/2014-1-30]]
 
*Comparison of FISSEQ_Adpt and Shifted FISSEQ_2ndRCAprimerDye in same wells (column 9): [[Matt:LabNotes/2014-1-31#Shifted_FISSEQ_vs_FISSEQ_Adpt_Signal_Count_in_Same_Wells]]

Latest revision as of 22:03, 6 February 2014

Checking non-specific binding of Shifted FISSEQ_2ndRCAprimerDye[edit]

  • 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[edit]

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[edit]

  • 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
    • This can be further explored by seeing of adding RT primer (which has complementary sequence to RCA primer site) can reduce hybridization of Shifted FISSEQ, which would indicated Shifted FISSEQ is hybridizing to the RCA primer sequence of primary rolonies: Matt:LabNotes/2014-1-30