Gel electrophoresis is a revolutionary technique used in molecular biology, genetics, and biochemistry for separation of RNA, DNA, and proteins based on the charge, size, and various physical properties. Naturally, for every researcher conducting experiments in the laboratory is aimed at optimizing the process. However, mistakes may still occur especially when new researchers handle the procedure.
Listed below are the common mistakes that one needs to avoid when conducting the gel electrophoresis procedure in the laboratory:
Not preparing the samples carefully
One of the most critical steps to perform during gel electrophoresis procedure is the preparation of samples. Now, not diluting the samples appropriately or using more samples than needed may cause distorted bands, lower the resolution, and overloaded wells. When preparing protein, DNA, or RNA samples, ensure that they are free from contaminants like ethanol, which may alter the result. Furthermore, you need to apply the correct buffer and mix the sample with the loading dye properly for the best results.
Choosing the wrong buffer
Choosing the wrong electrophoresis buffer may affect the separation of molecules, for which the experience is conducted. Equally important is to pay attention to the fact is the freshness of the buffer. For instance, TBE is more appropriate for smaller fragments and TAE is ideal for separating larger fragments of DNA. When starting the procedure, be sure to choose the correct buffer and prepare it according to the right protocol. Moreover, you need to be careful about when changing the composition of the buffer as it may affect the outcome of the procedure.
Not using quality samples
Suring sample preparation in gel electrophoresis, you need to check the quality of the sample used for this purpose. Remember that the quality of sample may lead to contamination, degradation or overloading. Furthermore, elements like heat, enzymes, or pH changes may result in low-intensity bands. That’s why you need to ensure that the samples are stored properly and fresh reagents used. It is the samples based on which the entire procedure takes place. So, unless it is prepared the right way, the gel electrophoresis may lead to inaccurate results.
Incorrect preparation of gel
When preparing the gel for electrophoresis procedure, make sure the purpose of conducting the experiment. The gel contains a polymer matrix for the creation of pores of different shapes and sizes, based on the concentration and the types of the polymer. Usually, polyacrylamide and agarose gels are suitable for different applications and molecules.
Now, not taking proper care with the concentration of the polymer may again impact the resolution and the size of the pores. As part of electrophoresis troubleshooting, you need to take care of the preparatory regime. From using the correct values of the molecule types to preventing the formation of air bubbles, and setting the temperature right, everything has role to play in this procedure.
Inaccurate gel casting
Inaccurate setup of gel or gel casting may also distort the results. That way, bubbles are likely to form when pursing the gel, resulting in inconsistent migration of molecules. So, when casting the gel, let it be free from bubbles.
Improper time and voltage considerations
The time for which the gel electrophoresis procedure takes place and its speed depends on time and voltage. Now, the voltage determines the movement of molecules in the gel and the time is how long the procedure is slated to continue. If you fail to run the procedure for insufficient time and apply wrong voltage, the sample may run far more speedily that is required or vice versa. Ideally, you need to adhere to the recommended voltage of the gel and the molecular size to obtain correct results. A common mistake is when the voltage is turned on too high or too low, which damages the molecules and the gel. Furthermore, it can lead to low resolution or diffusion. To avoid such instances, you need to track the current and temperature and allow the procedure to move smoothly.
Inadequate staining
Not staining the gel adequately may lead to inconclusive results and often too difficult to interpret. While excessive stain results in background interference, too less stain causes invisible or weak bands. So, make sure to stain your gel with a suitable dye for the right amount of time for accurate outcome.
Gel electrophoresis is a significant procedure that takes place in the laboratory. When you avoid the abovementioned mistakes, it helps improve the efficiency and accuracy of the experiments. Want to master the electrophoresis process? At IGene Labserve, we manufacture top-notch laboratory equipment for your experiments. Explore here https://www.igenels.com/ and order yours today.