“Common factors affecting the results of DNA agarose gel electrophoresis are electrical field, nucleic acid sample, buffer and other chemicals used which inversely influence the final results.”
Do you know?
- TBE is the common replacement for TAE- an electrophoresis buffer. However, which to use depends on the experiment.
- The EtBr- Ethidium Bromide, helps to visualize DNA and is carcinogenic, can cause cancer.
- BPB- bromophenol blue can’t be dissolved directly into the water.
- RNA migrates faster than the DNA in a gel.
Electrophoresis is an extensive subsidiary molecular genetic technique often used in genetic labs. Its applications are to separate nucleic acid- DNA/RNA, to study mutations, perform restriction digestion and separate PCR amplicons.
It is a technique in which charged particles such as DNA migrates under the influence of electrical current. Buffer provides media, BPB dye helps in monitoring the migration and EtBr helps to visualize under UV light. Notedly, these are also factors that adversely influence gel electrophoresis results.
In the present article, I will explain to you some factors that affect the results of gel electrophoresis or DNA agarose gel electrophoresis and how to overcome it. But before that if you are a newbie, this article may help you to understand the basics of the present topic:
Key Topics:
What are the factors that affect DNA agarose gel electrophoresis?
Here we have categorized and enlisted common factors that affect the results of agarose gel electrophoresis of DNA.
- Nucleic acid sample- Type, purity and quantity.
- Buffer- concentration and pH of buffer and buffer type.
- Electric field- voltage applied current and charge of particles.
- Other- gel preparation, gel concentration, other chemicals.
Type of sample:
Varied techniques of gel electrophoresis are used for protein, DNA and RNA separation. Though the basic technique of separation remains the same, minor changes are needed.
RNAs are smaller than the DNA, thus migrate faster than DNA in a gel. So to run an RNA gel, we need a gel with a different concentration (we are not talking about protein here).
The concentration of a gel for DNA and RNA varies. Sample type also has a significant effect on gel electrophoresis results. The concentration of gel, preparation technique, running time and other factors vary depending upon the sample types.
Purity of nucleic acid:
Mishandling and inappropriate DNA extraction cause serious problems in PCR and electrophoresis. To get good results, DNA should be nearly pure. A DNA with a 260/280 ratio of approximately 1.80 is highly recommended.
To run the gDNA, pure DNA migrates properly in a gel, contrary, the migration pattern of contaminated DNA isn’t interpretable.
Two common contaminants of DNA extraction are RNA and protein. A smear below DNA and a smear above DNA band are observed when get contaminated with protein and RNA, respectively. Pure DNA separates evenly, beautifully and distinguishably in a gel.
If you wish to achieve good yield and quality of DNA this article is for you: How to Increase DNA Purity and Yield?
Contaminated DNA gel is seen in the figure below,
Concentration of DNA:
Concentration also matters to separate DNA perfectly. The concentration of DNA and the size of gel pores have a relation! Larger DNA fragments can’t migrate efficiently from a smaller sized gel.
Therefore the concentration of DNA is important for separating DNA and getting good results. For example, to run genomic DNA we need 0.8% gel while to run PCR amplicons, we need 2.0% gel.
(can you tell me which percent gel is required for restriction digestion?)
Sometimes, highly concentrated DNA can’t separate on a gel, we need to dilute it, although diluting the sample decreases the concentration but not purity. Ideally, a 0.8% gDNA gel can separate 500 to 900μg DNA.
Type of buffer:
Buffer is an essential ingredient of the electrophoresis, as it provides a constant liquid medium and pH during the run. TAE and TBE are two common systems widely used during gel electrophoresis of the DNA.
However, both don’t have the same activity. A newbie can use either but for the specialized experiment or for critical investigations, the expert needs to choose the best from either.
The Borat can react with the sugar portion of the DNA and influence the migration, consequently and therefore not recommended by experts. Tris-acetate-EDTA is the first choice to run DNA, worldwide.
A pH of the buffer:
pH is as important as other factors, as minor changes in the pH of the buffer adversely influence the migration of DNA and gel results, consequently. pH 8.3 is ideal for running DNA perfectly.
Related article: Role of EtBr in Agarose Gel Electrophoresis and Karyotyping.
Composition of the buffer:
The chemical composition of TAE buffer is Tris, acetate and EDTA and the chemical composition of TBE buffer is Tris, borate and EDTA. Each component executes a vital role to control separation, so their concentration and quantity matter a lot!
We have covered an article on this topic. You can check out the chemical preparation here: Agarose gel electrophoresis buffer.
Change in composition causes slower migration, DNA degradation or other problems in gel electrophoresis. So you need to weigh and prepare each buffer correctly. Read the above article.
Current and voltage:
The current and voltage of the run affect the migration and separation of biomolecules. When the applied current is too high, it makes DNA run faster, heats the gel and buffer and makes DNA smear.
DNA can’t separate correctly.
When the applied current is too low, DNA can’t run from the gel pores and diffuses in a gel. Fragments of DNA can’t be separated in either case. Ideally, 80 to 100v current is advisable to run a DNA gel. Note that the voltage applied also depends on the size of DNA fragments.
The gDNA is run by 60 to 80V whilst PCR amplicons are run by 100 to 120V, ideally. Take the advice of an expert before deciding the current and voltage of the gel. The current of electrophoresis should also remain constant during the whole run to run DNA at a constant speed.
Read more: 10 Proven Tips to Success in Gel Electrophoresis of DNA.
The concentration of agarose:
Agarose is an ingredient of a DNA gel. It is boiled in the buffer to make it gel. Every DNA sample is different, a varied concentration of agarose is needed to run different samples.
(We have discussed this point somewhere in the article) to separate larger DNA molecules, low concentration gel whilst to separate smaller DNAs high concentration gel is recommended. Here is the table which helps you to understand the point:
|
Concentration of Agarose |
Types of DNA sample |
approx. fragment size |
|
0.8% |
Genomic DNA |
> 1 kb |
|
1.0% |
PCR product and plasmid DNA |
400bp – 10kb |
|
2.0% |
PCR product |
50bp – 2kb |
|
3.0% |
Restriction digestion |
10bp to 1000bp |
If the concentration of agarose and quantity of DNA can’t be maintained fragments can’t be separated efficiently.
Gel preparation:
Superior expertise is though not required to prepare a gel, gel preparation makes a huge difference. If the gel isn’t settled, having air bubbles, not evenly distributed and the thickness isn’t good, our DNA can’t migrate accurately.
Air bubbles stop DNA running, and is one of the most common gel preparation problems beginners do. One needs so much practice to do it perfectly.
Another factor is gel thickness and inappropriate distribution. A gel caster should be balanced first before pouring the gel. In addition to this, the quantity of gel must be sufficient enough too.
Evenly distributed gel having 1 to 1.5cm thickness is recommended. If the thickness is too low, DNA molecules can’t migrate at a constant rate.
Handling errors:
Performing experiments nearly perfect is critical too. Handling errors are so common, and that reflects in results, though only experts can find. Handling errors mentioned below are common.
- Inaccurate chemical preparation.
- Haphazard weighing and solution preparation
- Inaccurate pipetting
- Inaccurate pH balance
- Handling errors in DNA extraction
- Inaccurate sample preparation
Your hard work and accuracy reflect in your work, how you prepared and run a gel; therefore every step should be performed sincerely. Handling errors aren’t accepted for detection of mutation, validating experiments and disease diagnosis.
Pipetting chemicals and solution are yet another common mistakes that influence results of DNA gel electrophoresis. Learn correct pipetting technique, understand when to use double press and when to use the triple press. Besides, other factors are loading DNA samples in wells, inappropriate labeling of samples, preparing BPB dye, etc.
Conclusion:
DNA extraction, gel electrophoresis of DNA and PCR are three important techniques of the genetic lab. To pursue a career in genetics, a fellow should have to learn at least these three techniques precisely. The gel electrophoresis is not only used for research purposes but also used in diagnostic experiments as well.
We have covered serious articles on electrophoresis technique, which are mentioned in various links somewhere in this article. You can read it to learn. We also have explained accurate chemical preparations in individual articles. You can search article and read by clicking links giving somewhere in this article.
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