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*[[Hosuk:Lab_Notes|LabNote]] ===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==== *% 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===== *Pos 6 : for strip test {| {{table}} border = 1 | align="center" |'''Pos 6, 5uM''' | align="center" |'''Pos 1, 5uM''' | align="center" |'''Pos 2, 100nM''' |- | align="center" |'''Pos 5, 10nM''' | align="center" |'''Pos 4, 1nM''' | align="center" |'''Pos 3, 0.1nM''' |} =====Counting===== *px : 2~100, bkmulti = 13 {| {{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" | | align="center" | 0.1nM | align="center" | 1nM | align="center" | 10nM | align="center" | 100nM | align="center" | 5uM | align="center" | 5uM |- | align="center" | 1st Rolony || align="center" | 7387 || align="center" | 6495 || align="center" | 6040 || align="center" | 6089 || align="center" | 7054 || align="center" | 8663 |- | 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 |} {| {{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" | 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]]
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