You can measure the concentration of your DNA during cloning steps more precisely than NanoDrop by running the gel with 1 KB marker. You can use ThermoScientific GeneRuler 1 KB DNA marker. If you refer its standard protocol, you can find ng/0.5ug values.

Then just run your DNA sample with the ladder and match intensity of your DNA band with the bands in the ladder. Read the corresponding ng/0.5ug values of bands that will be your concentration of DNA in the sample.

Suppose concentration of your ladder is 0.1 ug/ul, then add 5ul ladder to get 0.5ug of the ladder. If you add 10 ul of DNA sample, compare its intensity with band in ladder and find the corresponding ng/0.5ul value, which gives you concentration in ng/10 ul of DNA sample.

https://www.google.co.in/search?q=thermo+generuler+1+kb+plus&espv=2&source=lnms&tbm=isch&sa=X&ved=0ahUKEwi1v4298fjNAhUDMY8KHfC5CbUQ_AUICCgB&biw=1366&bih=677#imgrc=3DfFp--g6RoeKM%3A

you can not quantitate your DNA on gel but there is a way to do it, but it not accurate

Hi Rasool,

Thank you very much for commenting.

I know that it is not 100% accurate. I do not trust the NanoDrop reading because at times it gives me absurd reading (especially when I am doing cloning). For instance, at times it it gives reading of my double digested gel purified vector as low as 0.2 ng/ul. This is so untrue because whenever I run the same sample on the gel (1.0 ul), it shows strong intensity on the gel.

So please do not rely nanodrop reading if it is showing anything less than 20ng/ul. If it is less than that, same sample usually show lot of variation. But I do not have any specific answer for your initial question.

Thank you Navonil.

I'll bear that in mind

Repeat the reading with the nano drop: clean it with sodium hypochlorite 0.5% then read a drop of ddH2O then tray to read your sample

use of Nanodrop is perfect solution

I also don't understand the problem in using a NanoDrop system. You blank it with the same elution liquid, you measure it and it's perfect. Did you measure your sample more than once? We also use a NanoQuant system from Tecan, based on the same principle (UV absorption) and it also produces reliable data.

Yes. My Institute has three different NanoDrop (one of it is pretty new). I tried repeating the reading using all of these Nanodrop, but it gave me similar reading.

When the all machine gave you smilar result that is mean that's correct

Dear Saranpal,

There is a kit from invitrogen to quantitate DNA.Its called Quant IT picogreen kit which uses a lambda calf thymus DNA as a standard.You can generate a standard curve based on the range of DNA you would expect and quantitate DNA accurately.

http://probes.invitrogen.com/media/pis/mp07581.pdf

Dear Saranpal,

if you want to quantify double strand DNA and exclude degraded or single stranded DNA (which is not excluded by NanoDrop technologies (atleast of what I know) I recommend for u to use the Qubit technology from Invitrogen.

http://de-de.invitrogen.com/site/de/de/home/brands/Product-Brand/Qubit.html?CID=fl-qubit

It is much more accurate than a NanoDrop. The only problem may be, that you will need a specific Fluorometer for the quatification. If money isnt an issue, I recommend using Qubit over NanoDrop in all quatification dependent manners and experiments.

Thanks for the suggestions. I think Quant it Picogreeen would be a better option for me to quantitate (cheaper). I did not know about it. I have always thought that quantification could only be performed using markers. Did not know that there is a specific dye that could help in quantifying. Thanks for the help guys

Dear Saranpal,

before you actually order any new assay equipment you may consider asking around in your research facility if anybody else may be interested in buying a new quatification system for DNA (e.g. Qubit , as I suggested above).

I have a short comment on the Picogreen assay. I havent worked with that assay myself yet, but from what I heard you have to run a standard (to create a regression curve) EVERY time you want to measure DNA.

As I said I havent worked with the Picogreen system before, but to me it sounds like this is only made for high throughput analyses when you have many different DNAs to measure.

Anyways, you should run with what you think is best for your purposes. Good luck for your quantification.

I think imageJ is quite capable of doing it. see the tutorial here

http://imagejdocu.tudor.lu/doku.php?id=video:analysis:gel_quantification_analysis

Concerning image J.

Seems like the cheapest solution for your problem.

But you need to keep in mind that performance of your gel electrophoresis is very important , if using image J. You will have to run a standard first, with defined DNA concentrations (so that you can actually compare the intensities) and you need to run all your gels under the same conditions and need to use the same setting when taking a picture (exposure time, % gain etc.). Otherwise results wont be comparable.

Also, if you know the concentration of your molecular weight standards, you can compare the intensity (EtBr and UV) of your concentrated and diluted samples with the closest migrating standard band (must have similar length in bps).

You can use several online software to do it (GenAnalyzer for example - Simple and fast). This program compare the pixel intensity you get from each band of your DNA ladder and compare it, with the help of a calibration curve, to the pixel intensity of your band.

Hi Saran

I used nano drop to quantify DNA and then confirm by agarose gel.

This is how I did it.

Prepare 1:1, 1:2, 1:4 dilutions of your DNA and quantify using a nanodrop. Make sure you do Normal and not serial dilution. Then you will need to run the above diluted DNA on agarose gel.

Based on the concentration values try to load the amount that corresponds to the ng of DNA for a selected band given in the standard quantitative ladder. Make sure you select the band from DNA ladder that is equivalent or close to the size of your sample DNA.

If your quantification is correct the intensity of bands in all your sampke dilution should be same or comparable to the intensity of band selected on your DNA ladder.

These bands should can be further quantified by any image analysis software like AiDA or ImageJ, but that is not necessary.

You can skip the dilutions step and carry quantification for undiluted sample, if you are working with large sample number.

Many many thanks guys for the suggestions.

And thanks Patrick and Izhar. Your suggestions have really been helpful thus far :-)

You can measure the concentration of your DNA during cloning steps more precisely than NanoDrop by running the gel with 1 KB marker. You can use ThermoScientific GeneRuler 1 KB DNA marker. If you refer its standard protocol, you can find ng/0.5ug values.

Then just run your DNA sample with the ladder and match intensity of your DNA band with the bands in the ladder. Read the corresponding ng/0.5ug values of bands that will be your concentration of DNA in the sample.

Suppose concentration of your ladder is 0.1 ug/ul, then add 5ul ladder to get 0.5ug of the ladder. If you add 10 ul of DNA sample, compare its intensity with band in ladder and find the corresponding ng/0.5ul value, which gives you concentration in ng/10 ul of DNA sample.

https://www.google.co.in/search?q=thermo+generuler+1+kb+plus&espv=2&source=lnms&tbm=isch&sa=X&ved=0ahUKEwi1v4298fjNAhUDMY8KHfC5CbUQ_AUICCgB&biw=1366&bih=677#imgrc=3DfFp--g6RoeKM%3A

The Nanodrop isnt the best solution in every case. If u want to quantify dsDNA you will overestimate the concentration if there is degrated DNA present. So using the gel instead of price intensive capilar electrophoresis systems is a good alternative. Also using things like the Qubit is price intensive and you dont get an information about the molecular weight.

Although its too late to answer, Saranpal Chhabra usually nanodrop cannot accurately quantify below concentrations of ~10 ng/µL. To quantify by gel electrophoresis refer the initial concentration of the DNA ladder and then you know the amount of DNA in each band.(amount of DNA of the ladder will be already given by the manufacturer).