How can I do agarose gel electrophoresis, and then compare the bands to the gene ladder and detect the concentration of the gene?
When you buy commercially available DNA ladders, often the also give the concentration based on the brightness of the band. You can compare the brightness intensity of your band with that of the corresponding band of the ladder and estimate the concentration to a very good approxiamation.
When you buy commercially available DNA ladders, often the also give the concentration based on the brightness of the band. You can compare the brightness intensity of your band with that of the corresponding band of the ladder and estimate the concentration to a very good approxiamation.
DNA concentration by agarose gel electrophoresis ideal than measuring with nanodrop or spectrophotometry.You can not only measure the concentration but also see the quality of DNA on gel.
all the best
Hello Zahra,
Do as follows:
1. Isolate your DNA from the source. Make sure that you do RNAase treatment during or after the DNA extraction (highly advisable)
2. Anlayze the purity of DNA at 260/ 280 nm (for RNA contamintaion) and 260/ 230 nm (for organice solvent conatmination)
3. Cast an agarose gel 1.8% --add EtBr just before casting the gel (when it is not very hot but still not solidified)
4. Load your sample using by bromophenol blue ( act as a loading dye and indicator). Remember the smaller the DNA fragment, the faster it moves. Also note that a coiled circular plasmid DNA will move faster than the nicked or linear DNA for the same reason.
5. Also load the ladder. The ladder depends on the size of DNA you are expecting. In general if your DNA source in Ecoli you can use 100bp-1000bp ladder. It is used to compare the length of size of your DNA using ladder as a reference scale.
6. Run at 60 mA, 75mV for 1-1.5hrs
7. Analyze under UV, find out your DNA size. Capture gel images.
Hope this helped.
Cheers!
Jitendra
Hi Zahra
As I know with 1% agarose gel electrophoresis you can be sure about DNA quality, but you can’t measure the quantity by this method.
Good luck
Zahra
I agree with Abhishek. But, Kasap et al (2006) could give you proper consideration.
Find out the article here.
Good luck!
http://dergipark.ulakbim.gov.tr/tbtkmedical/article/view/5000030810/5000031046
Agree with Abhishek and Danny gazali, You just need to compare the intensity of your band with the intensity of that lader/marker's band.
Good luck
As mentioned by Abhishek you can use commercial DNA ladders. Some gel viewing machines have software which can help you assess the quantity based on the brightness of the band with respect to the ladder. Syngene is one such company.
Be aware that this gives you very approximate quantitative results. It depends by what accurate means to your experiment. In classical PCR better quantification is obtained by doing a mimic PCR which employs a "competitor" of known concentration. Normally a competitor should differ by at least 50 bp in length in order to be able to easy discriminate it from the target. You run about 5 reactions in which you input your sample together with serial dilutions of the competitor. Run agarose gel and visualize results - looking for the band pair which seems similar in brightness. This method is tedious and competitor availability is a prerequisite. I used to do that by preparing it myself in the past. Also not very accurate, plus you need 5-10 reactions (not always you get the "final" result, could be an interval between 2 concentrations of the competitor, so you need to go further explore that interval). It's also costly. Gel imaging is a good suggestion but costs as well. The most accurate method is to set up a good qPCR reaction. Consider your purpose, your budget, and your time to decide one way or another
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