Agarose Gel Amount of DNA to Run?

[dave2006]

Hi, I am running a gel and was wondering how much DNA (is it in ng or ug?) we need to load in our gel?

I usually use 1 uL of loading dye diluted and 5 uL of my DNA sample and it usually works out ok .

However, how do we actually calculate the how much DNA in a PCR product or extraction to load on an agarose gel?

thanks

[molecule2005]

Hi,
its great to do that as its easy however yes it is not scientific at all especially if you want to publish.

When you run 5ul you have no ideas what concentration you have there of DNA. However, if you are running the DNA to determine the concentration by comparison to a Lambda ladder which has a specific quantity of DNA ladder thats another story...

Well, if you use ethidium bromide (EtBr) gels, you will need about 20ng of DNA to visualize it under UV light.

I would first measure your DNA concentration by UV spectrophotometry.

Then load about 25-100ng on your gel.

Note this is only if you are expecting only one band; You would have to multiply the 25-100ng with the number of bands if you do expect more than one band on your gel.

[dave2006]

thanks a lot for your help, is there a constant you need to multiply with the absorption value?

What wavelength do i use to measure DNA concentration?

[molecule2005]

Hey again,

DNA absorbs light of certain wavelength so that light of 260 nm passing 1 cm through DNA at 50 ug/ml concentration (in water) has an absorbance of 1.0.


Set your UV spectrometer to use light of 260 nm.

A negative control or blank cuvette (tube) is filled with water or just the buffer solution (such as TE; usually water) that the DNA is in. As the tube has no DNA, it should give an OD reading of 0. The machine however may show the reading to be something else. The machine is therefore 'zeroed' to set the reference at 0.

The sample to be analysed is diluted, usually 50-500 fold (usually 200-300 fold) in water (or buffer). The solution is puured into a similar-sized cuvette as the one used for blank/negative/reference and its OD measured.


The cuvettes used in Ultrospec 2000 have a 'path length' of 1 cm.


Multiplying OD (260 nm absorbance) with 50 ug/ml will give DNA concentration (inside the cuvette) and a further multiplication with the dilution factor (50-500) will give DNA concentration in the sample.

it is OD x 50, 260 Wavelength.

You measure at 260 but the ratios 260/280, 260/230 give you your purity.