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Daniel:Notebook/PosSequencing/2017-5-11
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=Bead Binding Test 2 (Started [[Daniel:Notebook/PosSequencing/2017-5-10|Yesterday]])= [[Daniel:Notebook/PosSequencing|Back to Calendar]] ==Image Processing== <ol> <li>Open new image in photoshop; use resolution 1344x1024 (same as .jpg images from microscope)</li> <li>Import brightfield and DAPI images to photoshop file</li> <li>Adjust levels as deemed useful using Image->Adjustments->Levels</li> <li>Create new red solid color layer</li> <li>Click on the mask icon (image next to red) and then go to Image->Apply Image...</li> <li>Use DAPI image as "Source" and choose "Multiply" under "Blending"</li> <li>Hide the original DAPI layer and save blended image</li> </ol> ==Results== <gallery perrow=2 heights=300px widths=300px caption="DAPI overlays for Sample 1 prior to melting"> File:Sample1-dsDNA-Overlay-F1-BlendAdj.png|Field 1 File:Sample1-dsDNA-Overlay-F2-Blend.png|Field 2 </gallery> <gallery perrow=3 heights=300px widths=300px caption="DAPI overlays for Sample 2 prior to melting"> File:Sample2-dsDNA-Overlay-F1-BlendAdj.png|Field 1 File:Sample2-dsDNA-Overlay-F2-BlendAdj.png|Field 2 File:Sample2-dsDNA-Overlay-F3-BlendAdj.png|Field 3 </gallery> ==Discussion== So, prior to melting neither one actually has any useful overlay between DAPI and the beads. So that sucks. Also, no point in analyzing the post-melting samples. I should repeat the experiment [[Daniel:Notebook/PosSequencing/2017-5-12|tomorrow]] using a no-DAPI sample as well to check the background DAPI signal from the beads. Although given the general lack of overlap this is not likely. Other alternatives include DRAQ5 or SYBR green, if we actually have some that is not in a qPCR mix. [[Category:PosSeq]] [[Category:20170508]]
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