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*[[Hosuk:Lab_Notes|LabNote]] ===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. {| {{table}} border = 0 | align="center" style="background:#f0f0f0;"|'''Pos6 : 0.1pM''' | align="center" style="background:#f0f0f0;"|'''Pos1 : 0.25pM''' | align="center" style="background:#f0f0f0;"|'''Pos2 : 1pM''' |- | align="center" style="background:#f0f0f0;"|'''Pos5 : 100pM''' | align="center" style="background:#f0f0f0;"|'''Pos4 : 10pM''' | align="center" style="background:#f0f0f0;"|'''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|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]] ===NCIH1975 Rolony : 1st try=== *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)==== *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 *[[Media:NCIH1975-20131202_S1_Step1_Pos2_G550.tif|Raw image of S1, Pos2]] [[File:NCIH1975_prelimResult_S1_Pos2.png|1200px]]
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