Agarose gel electrophoresis is an important technique in molecular genetics for a long. DNA bands can only be visualized using agarose gel electrophoresis. In genomic research, analyzing and interpreting the agarose gel electrophoresis results are very crucial.
A lot of expertise and experience are required for Interpreting gel electrophoresis results.
In this article, we are giving you a pictorial guide for analyzing and interpreting agarose gel electrophoresis results. We are explaining each type of electrophoresis results from worse to best. For doing this, we had performed several experiments in bad as well as in some good conditions.
So let’s start with some of the basics,
Factor affecting the gel electrophoresis results:
- The composition and concentration of the buffer
- The concentration of the agarose gel
- The purity and concentration of the DNA
- The voltage of the electrophoresis
- Use of the buffer and agarose gel
- Preparation of the gel
- The pH of the buffer and DNA
Protein and RNA contamination in gel electrophoresis results:
RNA molecules are lighter than the DNA. So, the RNA migrates faster than the DNA and DNA migrates faster than the protein See the Image,
Here, in the image, the smear above the DNA band indicates the contamination of the RNA while the smear below the DNA band indicates the contamination of the protein.
Read further on agarose gel electrophoresis:
- Agarose gel electrophoresis
- Agarose gel electrophoresis buffer
- DNA gel loading dye
- Role of EtBr in agarose gel electrophoresis
Agarose gel electrophoresis is used mostly for the gDNA analysis and for PCR result analysis. Here we are discussing the results of some of the gDNA gel and PCR product gel.
Now let’s start with the gDNA electrophoresis:
Analyzing and interpreting gDNA gel electrophoresis results:
See well 9: the DNA is trying to come out from the gel but not migrated properly. Also, the smear above the DNA is indicating the contamination of the RNA. All other DNAs are degraded.
From 64 to 79, in each well DNA is trying to come out of the well but some DNA remained inside the well. A couple of reasons are responsible for that Firstly, the wells are broken during sample loading (see 72, 74, 75, 76, 77, 78) and secondly, the air bubbles were formed during the gel casting.
One another possibility in this gel is that the comb is not placed correctly or the gel is disturbed during the removal of the comb. Because of these reasons, the gDNA is unable to come out from the well.
Furthermore, smearing above the DNA indicates the high contamination of RNA into the sample and smearing behind the DNA band (in the wells 75, 76, 77, 78, 79) indicates the contamination of protein.
Conclusively, the DNA is not extracted properly and the gel preparation is poor.
The DNA in wells 50, 51, 52, 53, 54 and 55 are degraded. The concentration of the DNA is very high in the well 59 hence it can not come out of the well. The comb is not removed properly from wells 56, 59,60, 61 and 62. The DNA samples are highly contaminated with proteins as well as RNAs (59 to 62).
The DNA in wells 31, 32, 33, 34 and 35 are highly contaminated with RNA. Now see the wells 37, 38, 39 and 40, the gel is not poured properly so the air bubbles have remained inside the wells which hindered DNA from migrating towards the positive pole.
Samples 45 and 47 are not extracted well, highly contaminated with protein and RNA.
Now, this case is a bit different from other gels. Here the gel loading buffer is reused so many times, therefore, the actual concentration of the buffer is changed during the electrophoresis of this gel. Due to this reason, the buffer hindered in the migration of DNA and smear of DNA band appeared.
Also, the gel is slightly brighter than other gels because of the fragments of other DNA (in each run some amount of DNA remains in the buffer which appears into the next run when we re-use it).
Read the next article of this series: Part 2: Analysing and Interpreting (Agarose) Gel Electrophoresis Results
Analyzing and Interpreting the gel results of PCR products:
let see some of the gel images of PCR fragments. 2% gel is required to separate PCR products because PCR products are the smaller fragments of DNA nearly ~100bp to ~1500bp.
Read Further on PCR,
- A Complete Guide of the Polymerase Chain Reaction
- What is a multiplex PCR?
- What is gene editing and CRISPR-CAS9?
The image is captured under the UV transilluminator instead of the gel doc system to show you the effect of EtBr on the gel electrophoresis results.
Here due to the re-use of a gel as well as the buffer, the EtBr is not properly spread into the gel. Further, the traces of the previous EtBr is also present in the gel. See the orange color near the wells, DNA and ladder these all are the EtBr molecules not spread well.
In this gel also the gel, as well as the buffer, reused so many times. See the condition of the bands. The comb is not placed/removed properly.
Now this image is pretty good but what is the problem?
Here the annealing temperature of the primer is not selected properly. So the primer is compromised with other complementary sequences present in the genome. The annealing temperature is too low in comparison with its actual annealing temperature.
Due to this reason, more than 4 bands of PCR amplicons are observed in the gel. Further, see the green arrow, a bubble hindered the separation of the DNA ladder.
Conclusively, the PCR is not performed with the optimum PCR conditions.
Now this gel is pretty good, isn’t it? DNA ladder is separated nicely and DNA is also appropriately amplified. But the concentration of the template DNA is a problem here.
The concentration of the template DNA used in this PCR reaction is very high. In a normal PCR reaction, 25 to 30ng concentration is sufficient. However, in this PCR reaction, the concentration of DNA will be more than 100ng.
The smear of the DNA along with the amplified product is observed due to this reason.
The shining dots in the gel are air bubbles. Due to the air bubbles, the ladder is not migrated properly see the first red arrow. Further, the ladder and DNA is too old, or not maintained properly, it’s degraded.
Now analyze this gel image, the DNA ladder ran faster than the samples. The samples are smeared as well which means that the buffer is too old, its concentration is altered or the pH of the buffer may be probably changed.
Remember, when we have the smears like this in any of the PCR products our buffer is the problem.
Now see the two red arrows, the bubbles which hinder the migration of the DNA.
We have seen all the types of DNA gel electrophoresis results and interpreted each type of electrophoresis results. But what qualities does a good quality electrophoresis gel has?
- Good and sharp bands
- Minimum primer-dimers
- A beautifully separated DNA ladder.
- No background or traces of other DNA in the gel
See the next gel image and analyze each parameter. Though the primer-dimers are present that is another issue. The result of the gel is beautiful and the bands are so clear and self-explanatory.
Some of the interesting articles:
- Real-time PCR: Principle, Procedure, Advantages, Limitations and Applications
- What is touchdown (TD)-PCR?
- What is Restriction Digestion and how to do it?
For achieving this type of good results you have to keep in mind of several points,
- Do not re-use the gel. If necessary use only twice.
- Do not reuse the buffer. If necessary use only twice or thrice.
- Prepare buffer freshly every time for the gel as well as the electrophoresis tank.
- Preserve DNA and DNA ladders properly in the cold chain.
- Use template DNA ~30ng to 50 ng not more than that, in the PCR reaction.
- Use only 10pMol primers. Do not use the PCR reagents more than given into the protocol.
- Use high-quality chemicals.
Read our next article in this series. The article is on the gel electrophoresis analysis of restriction digestion, cccDNA, linear DNA, supercoiled DNA and multiplex PCR: Part 2: Analysing and Interpreting (Agarose) Gel Electrophoresis Results.
Doing gel electrophoresis is though an easy task for an experienced person but guessing results need some more experience and technical knowledge.
I hope this article will boost your practical knowledge of gel electrophoresis and will help you in interpreting and analyzing gel electrophoresis results.
Comment below if any point is missing.