Daniel:Notebook/PosSequencing/2017-5-11: Difference between revisions

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>Djacobse
(Created page with "=Bead Binding Test 2 (Started Yesterday)= Back to Calendar ==Image Processing== <ol> <li>Open ...")
 
>Djacobse
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<gallery perrow=2 heights=300px widths=300px caption="DAPI overlays for Sample 1 prior to melting">
<gallery perrow=2 heights=300px widths=300px caption="DAPI overlays for Sample 1 prior to melting">
File:|Field
File:Sample1-dsDNA-Overlay-F1-BlendAdj.png|Field 1
File:Sample1-dsDNA-Overlay-F2-Blend.png|Field 2
File:Sample1-dsDNA-Overlay-F2-Blend.png|Field 2
<gallery>
</gallery>


<gallery perrow=3 heights=300px widths=300px caption="DAPI overlays for Sample 2 prior to melting">
<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-F1-BlendAdj.png|Field 1
File:Sample2-dsDNA-Overlay-F2-BlendAdj.png|Field 2
File:Sample2-dsDNA-Overlay-F2-BlendAdj.png|Field 2
File:|Field 3
File:Sample2-dsDNA-Overlay-F3-BlendAdj.png|Field 3
<gallery>
</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.

Revision as of 19:08, 11 May 2017

Bead Binding Test 2 (Started Yesterday)

Back to Calendar

Image Processing

  1. Open new image in photoshop; use resolution 1344x1024 (same as .jpg images from microscope)
  2. Import brightfield and DAPI images to photoshop file
  3. Adjust levels as deemed useful using Image->Adjustments->Levels
  4. Create new red solid color layer
  5. Click on the mask icon (image next to red) and then go to Image->Apply Image...
  6. Use DAPI image as "Source" and choose "Multiply" under "Blending"
  7. Hide the original DAPI layer and save blended image

Results

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 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.