I just run a marker with known amounts of marker bands. E.g. Hyperladder 1 has 100ng of dna in the 1000bp if you run 5ul. Just compare te intensity of your band to this either by eye of scan the image on any densitometry software. We use bio rad Quantity One.
There are several softwares available in this category. Please have a look at Gel Analyzer [1] , phoretix1D Pro [2] (trial usage), Fragment Analysis 1.1 , Image J and various other paid programs.
Gel Analyzer is free and looks easy to use.
[1] : http://www.gelanalyzer.com/
[2] : http://www.totallab.com/download/software/
ImageJ is a great option because it is a free open software written in Java at the NIH. It is a useful tool but sometimes not as user-friendly as other softwares.
You may find a quick and easy tutorial for agarose gel analysis at ImageJ Wiki:
http://imagejdocu.tudor.lu/doku.php?id=video:analysis:gel_quantification_analysis
And you may also use ImageJ for quantification of blots. For example:
http://rsb.info.nih.gov/ij/docs/examples/dot-blot/
All the best.
I really appreciate Mr Muhammad to put interesting question and the responders specially #Tiago
BTW you should keep in mind that this method is more an estimate than an exact measurement, because of many factors influencing this (e.g. not sufficiently pure samples, pipetting error, different running speeds at different gel sections (which is important since e.g. EtBr is also running on the gel and different DNA fragment sizes differ in dye load; to deal with this you should use more than one ladder per gel), different background fluorescence, different gel qualities (not homogeneous matrix), band smiling, possible overloading, possible secondary structure, etc...).
Anyway with good gels and samples and exact pipetting it can be exact enough for many topics.
I also use the technique like Mark mentioned. I wrote an Excel-sheet were I can put in the data of the marker whith known concentrations (intensity and also "center of mass" in ImageJ) to calculate the bp and concentration (linear regression for concentration and exponential regression for bp).
If you're interested in just let me know
Best Lukas
Hi Lukas, Thank you for sharing your method which sound interesting to me. I would be happy to learn how you do it in detail.
In genrell it is a quite simple technique.
You have to scan your blot with the bands you are interested in and a marker where you do know the bp and concentration. Concentration is often given as a percentage for each band (so you can calculate the single concentrations of each band).
When you analyze your blot in ImageJ you should be sure that you enabled the "Center of mass" (Analyse - Set Measurements) and increased the number of decimal-places to 9.
Then you define your your boxes arround the marker, press "1", move the box to the first sample, press "2" and on and on pressing "2" till your finished. Then press "3".
You'll get the histogramm in which you have to close the bottom of each peak using the "Straight"-Tool. With the wand you'll now get your measurements for each peak.
To get a Regressionline for the concentration you open Excel, choose the "Area-value" of each sample of the marker as x-value and the concentration you calculated as y-value. Calculating the linear regression line, get the formula y= x + b and calculate the concentration for your samples by using the measured area as x-value.
For the Size it is the same procedure but instead using the calculated area you'll use the x-mean and instead of the linear-regression you have to choose an exponential one.
Hopefully I could explain it clearly, if not let me know.
Keep in mind, this isn't an accurate method, so it would be the best to use different concentrations of your sample and maybe 2-3 markers on the left, right and middle of your gel.
Dear Lukas,
Thank you for complete explanation of Biorad Programs.
I downloaded the pro but it not work with jpg pictures, so what kind of extension should I save my pic to can analysis it by this pro. ??
Hi Mohamed,
I used ImageJ for the analyzing.
In generell I would prefer the *.tif format for all analyzing methods becouse there is no algorithm to minimize the size of the file. But it just make sense when you save the original file as tif (jpeg to tif doesn't increase the quality).
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