Hosuk:LabNotes/2013-11-21: Difference between revisions
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(10 intermediate revisions by the same user not shown) | |||
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====Result==== | ====Result==== | ||
* | *% of ppMALAT1 on 1st Rolony showed almost similar range along different concentration. | ||
*0.1nM of padlock probe seemed | *'''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. | ||
Line 63: | Line 63: | ||
| 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;"|'''Aligning''' | |||
| 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" | | |||
| 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:-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" | 49x49px || align="center" | 49x49px || align="center" | 49x49px || align="center" | 49x49px || align="center" | 49x49px || align="center" | 53x53px | |||
|- | |||
| 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" | 4614 || align="center" | 4060 || align="center" | 3691 || align="center" | 3999 || align="center" | 5911 || align="center" | 5321 | |||
|- | |- | ||
| 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" | 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]] | |||
=====Workflow===== | |||
*In order to match work area in both 1st rolony image and ppMALAT1 image after global aligning | |||
[[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
- Step 1 : Image 1st Rolony
- Step 2 : Strip, anneal ppMATLA1 (0.1nM, 1nM, 10nM, 100nM, and 5uM)
- 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