Hosuk:LabNotes/2013-11-21

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Revision as of 06:47, 22 November 2013 by >Hosuki78
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Padlock Probe Concentration Test

  • Sample : 96 well plates, made at 10/23
  • 2048 x 2048, 600Hz
  • Each stripping : 30min, and annealing : 15min
  • The concentration of the dye-labeled probe : 1uM
  • Use “Mark and Fine” function to save each location at each well


Procedure

  • Process Steps
    1. Step 1 : Image 1st Rolony
    2. Step 2 : Strip, anneal ppMATLA1 (0.1nM, 1nM, 10nM, 100nM, and 5uM)
    3. Step 3 : Add “Shifted FISSEQ_2ndRCAprimerDye” , image


Result

  • Need overlap counting, but
  • 0.1nM of padlock probe seemed many signal, and Need to plat toe…
  • Will do more further analysis


1st Rolony Count
  • Pos 6 : for strip test
Pos 6, 5uM Pos 1, 5uM Pos 2, 100nM
Pos 5, 10nM Pos 4, 1nM Pos 3, 0.1nM


Counting
  • px : 2~100, bkmulti = 13


Dye Probes Pos3 Pos4 Pos5 Pos2 Pos1 Pos6
0.1nM 1nM 10nM 100nM 5uM 5uM
1st Rolony 7387 6495 6040 6089 7054 8663
MALAT1 4886 4196 3817 4155 6179 5561
% 66.14 64.60 63.20 68.24 87.60 64.19


Dye Probes Pos3 Pos4 Pos5 Pos2 Pos1 Pos6
Aligning 0.1nM 1nM 10nM 100nM 5uM 5uM
offset x = -9, y = -43
Crop 51 by 51px
1st Rolony 6742
MALAT1 5528
Overlapped 4465
% 66.23



Workflow
  • In order to match work area in both 1st rolony image and ppMALAT1 image after global aligning

File:PadlockProbeConcTest Workflow1.png