AlanFung:LabNotes/Rolony/2013-9-26: Difference between revisions

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[[File:0711_SBS_1st_C03Cy3_Hist_Cutoff.png |left |thumb |300px |Cutoff at 5000]]
[[File:0711_SBS_1st_C03Cy3_Hist_Cutoff.png |left |thumb |300px |Cutoff at 5000]]
[[File:0711_SBS_1st_C03Cy3_Hist_Cutoff.png |left |thumb |300px |Cutoff at 5000]]
[[File:0711_SBS_1st_C03Cy3_Hist_Cutoff.png |left |thumb |300px |Cutoff at 5000]]
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<!--Start of the details for the top-of-table heading-->
|+style="background:transparent; color:black"|'''''The same table display, but with a white background, and five extra 25 pixel-wide columns, for spacing'''''
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|-align="center"
 
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|[[Image:0711_SBS_1st_C03Cy3_Hist_Cutoff.png|border|500x130px|none]]
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|[[Image:0711_SBS_1st_C03Cy3_Hist_Cutoff.png|border|500x130px|none]]
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|[[Image:0711_SBS_1st_C03Cy3_Hist_Cutoff.png|border|500x130px|none]]
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|[[Image:0711_SBS_1st_C03Cy3_Hist_Cutoff.png|border|500x130px|none]]
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<!--Start of a second row and add formats-->
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|width="25"|
|width="100"|'''Cutoff at 5000'''
|width="25"|
|width="100"|'''Cutoff at 5000'''
|width="25"|
|width="100"|'''Cutoff at 5000'''
|width="25"|
|width="100";;|'''Cutoff at 5000'''
|width="25"|
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Revision as of 00:29, 27 September 2013

Strip off dye coupling with formamide

  • Aspirate PBS from mattek dish with primary/secondary rolonies
  • Add 200ul preheated (65C-80C) 80% formamide in 2X SSC
  • Incubate 10min RT
  • Rinse w H2O
  • Add in 1:200-1uM 200ul (preheat 65C) ACTB-primary rolony target probe (Alexa 488) (200uM ACTB_1stRolonyFISH)
  • Incubate 10min RT
  • Rinse w 2x SSC twice
  • Add in 2X ssc
  • image

Matlab Image Analysis

Goal

  • The goal is to analyze the image taken from SBS sequencing of the primary rolony
  • I would create a threshold to screen out the noise
  • Call a base based on the signal area
  • Generate the 3 base sequence based on the location of the signal

Tools

  • Images (taken with Hosuk)

[1]

  • Matlab

Protocol

  • I picked the image as a test (cycle3 Cy3)
  • I=imread('sbs_cycle03_Cy3.tif') ;load the image
  • DI=double (I); Convert the image to a double class (when storing use single variable as it takes less memory, for processing convert variable to double)
  • whos ;Check that the variable indeed was converted into double
  • Hist(DI);
  • Looks like the cutoff is at 5000
The same table display, but with a white background, and five extra 25 pixel-wide columns, for spacing
Cutoff at 5000 Cutoff at 5000 Cutoff at 5000 Cutoff at 5000