The following are five different approaches to determining the quantity of DNA - Drawellanalytical.com

Comments · 47 Views

You have prepared your DNA, and you are now prepared to move on to the next stage of your experiment

You have prepared your DNA, and you are now prepared to move on to the next stage of your experiment. However, there is a good chance that after purification you won't find any trace of DNA in the final tube you examine. If you cannot visually inspect the DNA in your tube, how can you determine whether or not it is present?

There are a lot of different approaches to this, and the one you choose to take might depend on the instrument you have at your disposal, the amount of time you have, or both.

UV absorbance
One of the most common approaches to quantifying DNA is through the use of UV absorbance. In order to determine the concentration of substances within a liquid using this method, one measures the absorbance of light as well as its transmission through the liquid. Many different wavelengths of light are absorbed to varying degrees by molecules, and many molecules have a particular wavelength that is the one at which they absorb the most light. These absorbances are measured by the spectrophotometer using cuvettes that are transparent to UV light. The absorbance of the buffer that the DNA is in is the first thing that is measured. This is referred to as a "blank," and it measures the absorbance of the background. After that, you determine the DNA sample's absorbance by measuring it.

You can get a general idea of the concentration of your DNA preparation and whether or not there are any other contaminants from these absorbance measurements. At a wavelength of 260 nanometers, the absorption of nucleic acids (DNA and RNA) is at its peak. Proteins, on the other hand, absorb light most effectively at a wavelength of 280 nm, while organic compounds and chaotropic salts absorb light most effectively at a wavelength of 230 nm. As an indicator of DNA's degree of purity, the A260/A280 ratio is utilized. This number ought to ideally fall somewhere in the range of 1.8 and 2.0. The ideal ratio of A260 to A230 is one that is greater than 1.5.

 

 

After that, you are able to compute the DNA concentration by making use of the A260 reading. In general, an A260 value of 1.0 corresponds to an amount of pure dsDNA that is 50 ug/ml. To calculate an estimate of your DNA, use the following formula:

Calculating concentration (in ug/ml): A260 reading multiplied by dilution factor multiplied by 50 ug/ml

This approach is not only quick and easy, but it also does not call for any specific reagents. On the other hand, its sensitivity is limited when dealing with low concentrations of DNA, and it is unable to differentiate between DNA and RNA.

 


Fluorescence dyes
Utilizing fluorescent dyes that fluoresce when bound to DNA is yet another method that can be utilized to quantify DNA. The most important difference between these two approaches is that spectrophotometric methods measure all nucleic acids, whereas dyes like PicoGreen and SYBRGreen only detect double-stranded DNA. Examples of such dyes include PicoGreen and SYBRGreen. These methods are frequently used to quantify DNA for next generation sequencing because they are more sensitive than UV absorbance, particularly when you anticipate low concentrations in your samples.

  • In contrast to methods that are based on absorbance, methods that are based on fluorescence require a standard curve, which is a collection of samples that each have a known DNA quantity and their corresponding fluorescence

  • In this way, you will be able to quantify your DNA preparation by comparing the fluorescence of your sample to the curve provided

  • Even though setting up this method in the laboratory will take more time, many fluorometers will automatically calculate the sample concentration for you, so you won't have to worry about doing the math yourself

     

Agarose gel electrophoresis
The agarose gel method is not the quickest way to quantify DNA, but Laboratory Equipment Supplier is a method that can be used to not only determine how much DNA you have, but also determine whether or not your DNA is complete and whether or not it is the appropriate size. Methods that are based on absorbance are unable to provide this information to you, and you do not require a spectrophotometer or a fluorometer in order to quantify DNA using this method.

First, choose a DNA ladder with known concentrations and pour your gel containing a DNA intercalating dye (for example, ethidium bromide). Next, run your gel through an electrophoresis machine. The concentration of each band in the ladder is indicated on the packaging of many commercially available DNA ladders. After that, you run samples of your DNA at varying concentrations and quantify the results based on the band intensities of your DNA in comparison to the intensity of the ladder. It is best to make your comparison based on the band intensity of the fragment in the ladder that has a size that is most comparable to the size of your DNA sample. This technique works best for analyzing DNA fragments, such as those produced by PCR. This method also provides an indicator of DNA or RNA contamination based on the presence of other bands or streaking. This indicator is based on the fact that this method separates the bands. This method, in contrast to those based on absorbance, does not provide any information regarding the presence of chaotropic salts or contaminating proteins.

Electrophoresis in a capillary system
The process of quantifying DNA through the use of capillary electrophoresis is very similar to the process of quantifying DNA through the use of gel electrophoresis. Except that it's a lot more compact. And through automation.

In contrast to gel electrophoresis, this method only requires 1-2 ul of sample, and the run time for each sample is only a few minutes at most. During the run, DNA fragments are separated using electrophoresis while moving through a microfluidic or nanofluidic channel. Smaller fragments migrate quicker than larger fragments. A fluorescent dye that intercalates into the DNA is used to measure the size of these fragments over time. The dye is designed in such a way that it measures the smaller fragments first, followed by the larger fragments.

Although this technique is utilized frequently before next-generation sequencing or microarray research, it is not utilized nearly as frequently for the preparation of standard plasmids.

The method of diphenylamine
Diphenylamine is used in yet another absorbance-based method for determining the quantity of DNA. Diphenylamine, when exposed to acidic conditions, reacts with deoxyribose sugars to produce a blue complex that can be measured at a wavelength of 595 nm.

Because of its low sensitivity and lengthy execution time, this technique is not employed very frequently. Nevertheless, the measurements are taken in the visible range, and in the event that no other instruments are available, a standard ELISA reader can be used to interpret the results.

Some last reminders about quantifying DNA
When deciding on a method for DNA quantification, there are many factors to take into consideration, including cost, time, equipment, and the expected DNA concentration. It is essential to think about the benefits and drawbacks of each method, as well as the circumstances in which one approach might be more appropriate than another.



Comments