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

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====Result====
====Result====
*Need overlap counting, but
*% of ppMALAT1 on 1st Rolony showed almost similar range along different concentration.
*0.1nM of padlock probe seemed many signal, and Need to plat toe…
*'''0.1nM of padlock probe seemed still higher than the number of targets,'''
*Will do more further analysis
*So we need to try lower concentration than 0.1nM, such as 1pM, 10pM.




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{| {{table}} border = 1
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Dye Probes'''
| align="center" style="background:#f0f0f0;"|'''Aligning'''
| align="center" style="background:#f0f0f0;"|'''Pos3'''
| align="center" style="background:#f0f0f0;"|'''Pos3'''
| align="center" style="background:#f0f0f0;"|'''Pos4'''
| align="center" style="background:#f0f0f0;"|'''Pos4'''
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| align="center" style="background:#f0f0f0;"|'''Pos6'''
| align="center" style="background:#f0f0f0;"|'''Pos6'''
|-
|-
| align="center" | Aligning
| align="center" |  
| align="center" | 0.1nM
| align="center" | 0.1nM
| align="center" | 1nM
| align="center" | 1nM
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| 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" | offset || align="center" | x:-1, y:-47 || align="center" | x:-7, y:-47 || align="center" | x:-20, y:-47 || align="center" | x:-2, y:-42 || align="center" | x:-9, y:-43 || align="center" | x:-29, y:-52
|-
|-
| align="center" | Crop || align="center" | 51 by 51px  || align="center" | || align="center" | || align="center" | || align="center" |  || align="center" |  
| align="center" | Crop || align="center" | 49x49px || align="center" | 49x49px || align="center" | 49x49px || align="center" | 49x49px || align="center" | 49x49px || align="center" | 53x53px
|-
|-
| align="center" | 1st Rolony || align="center" | 6742 || align="center" | || align="center" | || align="center" | || align="center" | || align="center" |  
| align="center" | 1st Rolony || align="center" | 7001 || align="center" | 6264 || align="center" | 5777 || align="center" | 5851 || align="center" | 6753 || align="center" | 8257
|-
|-
| align="center" | MALAT1 || align="center" | 5528 || align="center" | || align="center" | || align="center" | || align="center" |  || align="center" |  
| align="center" | MALAT1 || align="center" | 4614 || align="center" | 4060 || align="center" | 3691 || align="center" | 3999 || align="center" | 5911 || align="center" | 5321
|-
|-
| align="center" | Overlapped || align="center" | 4465 || align="center" | || align="center" | || align="center" | || align="center" | || align="center" |  
| align="center" | Overlapped || align="center" | 4048 || align="center" | 3444 || align="center" | 3174 || align="center" | 3289 || align="center" | 4768 || align="center" | 4512
|-
|-
| align="center" | % || align="center" | 66.23 || align="center" | || align="center" | || align="center" | || align="center" | || align="center" |  
| align="center" | % || align="center" | 57.82 || align="center" | 54.98 || align="center" | 54.94 || align="center" | 54.64 || align="center" | 70.61 || align="center" | 54.64
|-
|-
|}
|}


* ppMALAT1 / 1st Rololy, after align, crop, and recount
[[File:Chart1_PadlockProbeConcTest.png|700px]]




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


[[File:PadlockProbeConcTest_Workflow1.png|700px]]
[[File:PadlockProbeConcTest_Workflow1b.png|650px]]

Latest revision as of 20:31, 22 November 2013


Padlock Probe Concentration Test[edit]

  • 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[edit]

  • 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[edit]

  • % of ppMALAT1 on 1st Rolony showed almost similar range along different concentration.
  • 0.1nM of padlock probe seemed still higher than the number of targets,
  • So we need to try lower concentration than 0.1nM, such as 1pM, 10pM.


1st Rolony Count[edit]
  • 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[edit]
  • 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


Aligning Pos3 Pos4 Pos5 Pos2 Pos1 Pos6
0.1nM 1nM 10nM 100nM 5uM 5uM
offset x:-1, y:-47 x:-7, y:-47 x:-20, y:-47 x:-2, y:-42 x:-9, y:-43 x:-29, y:-52
Crop 49x49px 49x49px 49x49px 49x49px 49x49px 53x53px
1st Rolony 7001 6264 5777 5851 6753 8257
MALAT1 4614 4060 3691 3999 5911 5321
Overlapped 4048 3444 3174 3289 4768 4512
% 57.82 54.98 54.94 54.64 70.61 54.64


  • ppMALAT1 / 1st Rololy, after align, crop, and recount

File:Chart1 PadlockProbeConcTest.png


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

File:PadlockProbeConcTest Workflow1b.png