Hosuk:LabNotes/2013-11-21: Difference between revisions

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| align="center" | MALAT1 || align="center" | 4886 || align="center" | 4196 || align="center" | 3817 || align="center" | 4155 || align="center" | 6179 || align="center" | 5561
| align="center" | MALAT1 || align="center" | 4886 || align="center" | 4196 || align="center" | 3817 || align="center" | 4155 || align="center" | 6179 || align="center" | 5561
|-
|-
| align="center" | % || align="center" | 66.14 || align="center" | 64.60 || align="center" | 63.20 || align="center" | 68.24 || align="center" | 87.60 || align="center" | 64.19
| align="center" | % || align="center" | 66.14 || align="center" | 64.60 || align="center" | 63.20 || align="center" | 68.24 || align="center" | 87.60 || align="center" | 64.19
|}
 
 
 
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Dye Probes'''
| align="center" style="background:#f0f0f0;"|'''Pos3'''
| align="center" style="background:#f0f0f0;"|'''Pos4'''
| align="center" style="background:#f0f0f0;"|'''Pos5'''
| align="center" style="background:#f0f0f0;"|'''Pos2'''
| align="center" style="background:#f0f0f0;"|'''Pos1'''
| align="center" style="background:#f0f0f0;"|'''Pos6'''
|-
| align="center" | Aligning
| align="center" | 0.1nM
| align="center" | 1nM
| align="center" | 10nM
| align="center" | 100nM
| align="center" | 5uM
| align="center" | 5uM
|-
| align="center" | offset || align="center" | x = -9, y = -43 || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |
|-
| align="center" | Crop || align="center" | 51 by 51px  || align="center" | || align="center" |  || align="center" |  || align="center" |  || align="center" |
|-
| align="center" | 1st Rolony || align="center" | 6742 || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |
|-
| align="center" | MALAT1 || align="center" | 5528 || align="center" | || align="center" |  || align="center" |  || align="center" |  || align="center" |
|-
| align="center" | Overlapped || align="center" | 4465 || align="center" | || align="center" |  || align="center" |  || align="center" |  || align="center" |
|-
|-
| align="center" | Overlap || align="center" |   || align="center" |   || align="center" |   || align="center" |   || align="center" |   || align="center" |
| align="center" | % || align="center" | 66.23 || align="center" | || align="center" | || align="center" | || align="center" | || align="center" |  
|-
|-
| align="center" | % || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" |  || align="center" | 
|}
|}





Revision as of 06:47, 22 November 2013


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