I am doing DNA electrophoresis. I wonder is there a way to quantify the amount of DNA in each band
I wouldnt use any software for quantifying DNA. I think that you can use it in a qualitative way though. There are a lot of potential biases when using such software and from what I was told and have done myself is to actualy extract each band, clean the product for instacne with a promega kit and then nano-drop it in order to get the exact concentration. I might be wrong though so lets see what others say. Good luck.
You can do an analytical curve in a 1D gel, with known amounts of BSA for example, use photoshop to quantify the pixels (the curve would be pixels x protein mass you applied for each well) and then you count the number of pixels in your sample bands and then plot them in the curve, I´ve done that with quite good results, you can find tutorials on the internet! But it´s quite time consuming... :)
If you need an estimation, you can use your ladder bands as a point of reference.
You know the concentration of each band in the ladder, so measure it's intensity (almost every graphical software can do that, but I recommend ImageJ), then measure your band and compare (don't forget to insert into your calculations correction for the difference in volume, if any).
Try using bands from your ladder that are similar to your band of interest.
Hello Yulia, do you mean the molecular weight standards? Where do I get the info regarding concentrations of each separate protein band of the MW standard? I use Biorad dual color standards, and couldn´t find the info anywhere! :)
below link provides the information about band quantification
http://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&ved=0CDcQFjAD&url=http%3A%2F%2Fwww.biostep.de%2FDocumentDownloadServlet%3Fdocid%3D17&ei=eBGIUOqEEcnMrQenvYHgCg&usg=AFQjCNGMeH478BcWANAY2D0UV4ZRxwfuyA
You can use a ladder as a refrence by comparison of each bands,the similarity between your ladder and bands can help you to measure them.
Like Jozef Nissimov says Nanodrop will give you an exact reading on the amount of DNA, however gel extraction kits have size restrictions. Have that in mind if you want to extract DNA from gels. You could lose DNA material.
Yulia and Rachel, i may be wrong but the intensity of bands probebly depends not just on concentration of the oligonucleotide, but also on length of oligonucleotide, as shorter oligonucleotides would bind to lesser number of Ethidium Bromide molecules. If this is the case, then some correction may have to be introduced for it.
That´s what I was about to say, regular protein electrophoresis shouldn´t be a problem I think! :)
Yadav, in case of DNA, length will influence the strength of the signal. but if you take as reference band that is in similar length (ladder band of 3Kb when your fragment is 3.3Kb, for example), the estimation will be quite good.
Hi Rachel,
The original question talked about DNA gels.
Regarding proteins - I looked at the info regarding your ladder, and they really does not provide quantity information.
What you can do is make as BSA standard curve and align your samples to it.
If they did not provide the size ref.. can you change to new DNA ladder that have size ref.??
hi yanfei li
i will suggest u to use the DNA ladder to compare the lanes during software analysis to calculate the band percentage or calculate the intensity based on peak height and peak area or during image capture increase the dpi (resolution) more than 100dpi images or simply pick the band from gel elute it with extraction buffer and go for estimation.
Maybe this will help:
http://sourceforge.net/projects/speedyquant
It compares gel bands in the same gel and calculates the relative amount (mass rather than concentration) of DNA. Yielded good results when tested with a DNA ladder.
hmm... works fine when i click it. But you will find it easily when you do google search with "SpeedyQuant" You can also have a look at the suppliers homepage for more information and some documentation: www.foreach-bioinformatics.de
Gelbuddy is most effective software.Just you have to install java on your computer (http://java.com/en/download/index.jsp) and than java advance image library (http://www.oracle.com/technetwork/java/current-142188.html) and download Gelbuddy (http://www.proweb.org/gelbuddy/). Just click on the Gelbuddy and enjoy yourself using it. Don't hesitate to ask for favour!
I suggest you try also http://www.gelanalyzer.com/
if you still need a solution.
Use gene ruler from fermentas each band tells concenteration you can calculate accordingly
image J, photoshop, they're pretty much all the same, you count pixels and can then reach an approximate value for quantifyingyour bands in comparison to a known standard. I did BSA standards ranging from 5 to 30 ug (silver nitrate staining, if you stain by coomassie you should double or triple these values for 13 cm gels), and then added my samples to the other gel slots. When I scanned the gel, I just counted the pixels for each standard, plotted a standard curve of pixels x concentration and voilá! Here: http://support.dalton.missouri.edu/index.php/wiki/Public:Quantifying_Color_Intensity/
This is a very easy tutorial, you can easily do this on photoshop!
Cheers! :)
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