Hosuk:LabNotes/2013-11-21: Difference between revisions
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| align="center" | 5uM | | align="center" | 5uM | ||
|- | |- | ||
| align="center" | offset || 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" | | | 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" | | | 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" | | | 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" | | | 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" | | | 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 | ||
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|} | |} | ||
[[File:Chart1_PadlockProbeConcTest.png|500px]] | |||
Revision as of 20:27, 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
- 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
- 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 |
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 |
File:Chart1 PadlockProbeConcTest.png
Workflow
- In order to match work area in both 1st rolony image and ppMALAT1 image after global aligning