Hosuk:LabNotes/2013-12-3: Difference between revisions
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=====0.1pM, 0.25pM ppMALAT1===== | =====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… | *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. | *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| | [[File:PPConcTest_LowerConc_Table1.png|700px]] | ||
*[[Media:PPConcTest20131202_96well-20131023_Step1_Pos6-G530.tif|Raw image of 1st Rolony on Position 6]] | |||
*[[Media:PPConcTest20131202_96well-20131023_Step2_Pos6-G580.tif|Raw image of 0.1pM ppMALAT1 on Position 6]] | |||
[[File:PPConcTest_prelimResult_0.1pM.png|900px]] | |||
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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) | *Image Cy5-1st Rolony detection probe (1uM in 30% Formamide + 2xSSC) | ||
====Result==== | ====Result(Preliminary)==== | ||
*The number of rolonies were not many, not as dense as that from PGP1F cells. | *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. | *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). | *'''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?... | **Do I need to analyze 3D-Rolonies along z-stack?... | ||
*Further analysis is on going. | *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 | |||
*[[Media:NCIH1975-20131202_S1_Step1_Pos2_G550.tif|Raw image of S1, Pos2]] | |||
[[File:NCIH1975_prelimResult_S1_Pos2.png|1200px]] |
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