NIH Image and electrophoretic gel analysis Date NIH Image and electrophoretic gel analysis

Emir ekhatipo at NOSPAMmidway.uchicago.edu
Fri Jan 19 18:18:20 EST 2001


Since you are writing about calibration data, I assume you are running
samples of control DNA with known concentration on the same gel as your
unknown samples, and you make sure that EtBr stains most of your DNA.

I myself would find it more convenient (and correct!) to use a "Measure"
macro, in particular its "Measure Sum of Pixel Values" function.
Most correct results will be obtained when your adjust the camera and
brightness/contrast settings so that the background is ~ zero pixels (or 255
in case of invert picture, since pixels can have values no more than 256 =
2^8 bits). After grabbing the picture into a file, use freehand or
"semi"-freehand select tool to outline the spot. Select the macro function
from the Special menu ("Measure Sum of Pixel Values"), press Ctrl-1 (= Do
Measure) and read the calculated values of mean pixel value and number of
pixels. One magnified by another gives you sum of all pixels (mean = sum of
pixels / number of pixels), which is equivalent to the volume of the peak =
quantity of DNA in the spot. As you understand, it is not necessary to be
very careful in outlining the band - take as much the area adjacent to the
spot as reasonable - it would not contribute to the final value of the sum
of pixels as soon as the value of background pixels ~ 0. If it is very
difficult to adjust settings so that the background is 0, subtract the
measures mean value of the background from the whole scan before doing
measurements.

Hope this helps.
Emir

"mcalexp" <mcalexp at my-deja.com> wrote in message
news:947uq9$4q0$1 at nnrp1.deja.com...
> Hello everyone,
>
>          I am new to using NIH Image so this might
>  be a silly question. I want to use Image to
>  quantify the amount of DNA in some samples.  I
>  fit the calibration data to a Rodbard-type curve
>  and then use the gelplot macro to mark the lanes
>  and obtain areas for the bands of interest.  Is
>  there a way for NIH Image to convert these areas
>  to the units used for calibration (e.g., ng DNA)
>  or are the gelplot macros used only for
>  determining ratios?  Is there a macro that would
>  let you calibrate using the areas (instead of the
>  8-bit gray-scale values) under the peaks of the
>  bands used for calibration?  Is it accurate enough
>  to use the gray-scale value (instead of the area)
>  of the band in question along with the calibration
>  curve to determine the amount of DNA (or whatever
>  else one is trying to quantify)?
>
>          Any help would be appreciated.  Thank you
>  all for your time.
>
>  Sincerely,
>  Alex
>
>
>
> Sent via Deja.com
> http://www.deja.com/







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