AlanFung:LabNotes/Rolony/2013-9-26: Difference between revisions
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*Looks like the cutoff is at 5000 | *Looks like the cutoff is at 5000 | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | ||
|- style="font-size:12pt" valign="bottom" | |- style="font-size:12pt" valign="bottom" | ||
| width="73" height="15" | Before Cutoff | | width="73" height="15" | Before Cutoff | ||
| width="475" | Histogram | |||
| width="71" | Binary Image | |||
|- style="font-size:12pt" valign="bottom" | |||
| height="15" | x | |||
| http://genome-tech.ucsd.edu/LabNotes/index.php/File:0711_SBS_1st_C03Cy3_Hist_Cutoff.png | |||
| x | |||
|} | |} |
Revision as of 23:56, 26 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)
- 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
Before Cutoff | Histogram | Binary Image |
x | http://genome-tech.ucsd.edu/LabNotes/index.php/File:0711_SBS_1st_C03Cy3_Hist_Cutoff.png | x |