Hosuk:LabNotes/2013-12-3: Difference between revisions

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===NCIH1975 Rolony : 1st try===
===NCIH1975 Rolony : 1st try===
*Sample : Glass-bottom MatTek dish – 2 dishes
*Sample : Glass-bottom MatTek dish – 2 dishes
*NCIH1975 P25
*NCIH1975 P25 (from Illumina)
*Process 11/30~12/02
*Process 11/30~12/02
*Image Cy5-1st Rolony  detection probe (1uM in 30% Formamide + 2xSSC)
*Image Cy5-1st Rolony  detection probe (1uM in 30% Formamide + 2xSSC)

Latest revision as of 00:19, 4 December 2013


Padlock Probe Concentration Test – More lower concentration[edit]

  • Sample : 96 well plates, made at 10/23
  • Padlock probe : ppMALAT1
  • Concentration at each well -->
    • Pos6:0.1pM, Pos1:0.25pM, Pos2:1pM, Pos3:5pM, Pos4:10pM, Pos5:100pM


  • Process Step
  • Step 1 : Image 1st Rolony (Cy5)
  • Step 2 : Strip with pre-heated (at 80C) 80% Formamide, incubate plate at room temp. for 30min.
  • Step 3 : Pre-heated 50uL ppMALAT1 at 80C, add ppMATLAT1 in each well, incubate plate at 50C for 30min.
  • Step 4 : Add 50uL of 1uM Shifted FISSEQ_2ndRCAprimerDye(in 2x SSC), incubate at room temp. for 15min.
  • Step 5 : Image
    • use two PMT Gain : (470 or 500) OR ( 500 or 530 ) OR ( 550 or 580 ) to get a similar intensity in the image, I’ll compare the number of rolonies in each PMT gain and decide which one is optimal gain.


Pos6 : 0.1pM Pos1 : 0.25pM Pos2 : 1pM
Pos5 : 100pM Pos4 : 10pM Pos3 : 5pM


Result (Preliminary)[edit]

  • FL signal was observed from the sample with 0.1pM ppMALAT1.
  • The observed ppMALAT1 seemed decreased along lower concentration of ppMALAT1.
  • Further analysis is on going.


0.1pM, 0.25pM ppMALAT1[edit]
  • Ratio looked very high… need to check the parameters are correct, maybe both 1st rolony and MALAT1 are over-counted…
  • The signal intensity from ppMALAT1 seemed weaker than that from 1st Rolony, but the number was not significantly decreased.
    • I think maybe the number (the concentration) of ppMALAT1 is lower than the number of target in a rolony...
  • However, the signal from ppMALAT1 looked more than I thought.
  • Will do further analysis


File:PPConcTest LowerConc Table1.png

File:PPConcTest prelimResult 0.1pM.png



NCIH1975 Rolony : 1st try[edit]

  • Sample : Glass-bottom MatTek dish – 2 dishes
  • NCIH1975 P25 (from Illumina)
  • Process 11/30~12/02
  • Image Cy5-1st Rolony detection probe (1uM in 30% Formamide + 2xSSC)


Result(Preliminary)[edit]

  • The number of rolonies were not many, not as dense as that from PGP1F cells.
    • --> However the rolony/cell is counted ~200 which looks fine.
  • Cells were not dense, it maybe reason why there is lower density of rolonies, need to wait 2~3 days more to get denser cells.
  • The thickness for z-stack is about 50~60um which is 2~3 times thicker than PGP1F (15~20um).
    • Do I need to analyze 3D-Rolonies along z-stack?...
  • Further analysis is on going.


Number of Rolony[edit]
  • S1, position 2
  • Laser Power = 4%
  • PMT Gain = 550 --> Count = 6311
  • Cells = ~30
  • Rolony/Cell = 210.37

File:NCIH1975 prelimResult S1 Pos2.png