Hosuk:LabNotes/2013-12-3

From ZhangLabWiki
Revision as of 18:42, 3 December 2013 by >Hosuki78 (Created page with "*LabNote ===Padlock Probe Concentration Test – more lower=== *Sample : 96 well plates, made at 10/23 *Padlock probe : ppMALAT1 *Concentration at each ...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search


Padlock Probe Concentration Test – more lower

  • 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

  • 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.



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

  • The number of rolonies were not many, not as dense as that from PGP1F 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).
  • Do I need to analyze 3D-Rolonies along z-stack?...