Hosuk:LabNotes/2013-12-3
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Padlock Probe Concentration Test – More lower concentration
- 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)
- 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
- 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
- Sample : Glass-bottom MatTek dish – 2 dishes
- NCIH1975 P25
- Process 11/30~12/02
- Image Cy5-1st Rolony detection probe (1uM)
Result(Preliminary)
- 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
- S1, position 2
- Laser Power = 4%
- PMT Gain = 550 --> Count = 6311
- Cells = ~30
- Rolony/Cell = 210.37