A buffer is an important ingredient in any biological reaction. “A biological buffer solution is a combination of a weak acid and conjugated base or vice verse which helps in maintaining constant pH of the solution”.
Tris-borate EDTA and Tris-acetate EDTA are the two most common types of buffer solutions used in agarose gel electrophoresis of DNA. To understand the role of the electrophoresis buffer, we have to understand the importance of electrophoresis and each component used in electrophoresis.
Electrophoresis separates DNA/RNA or protein molecules on the basis of molecular weight and a net charge of the molecule. This will helps in identification and characterization of the molecules.
Electrophoresis buffer, agarose, EtBr and DNA gel loading dye are an important component of agarose gel electrophoresis. We have written an article on EtBr and DNA gel loading dye separately. Read it here,
In this article, we will discuss gel electrophoresis buffer, the importance of gel electrophoresis buffer, the role of TAE and TBE buffer in agarose gel electrophoresis, preparation of 10X TAE/ TBE buffer and about my ultimate guide of using agarose gel electrophoresis buffer.
Importance of gel electrophoresis buffer
Two important functions performed by gel electrophoresis buffer,
- It maintains the pH of reaction nearly neutral. The weak acid and base in buffer keep pH in the desired range.
- By maintaining neutral pH, it controls the net charge of molecules which helps in proper migration and separation of the molecule.
Further, the DNA gel electrophoresis buffer prevents DNA from DNase attack. It also prevents the hydrolysis of DNA molecules by providing a constant liquid medium.
Even the liquid medium controls the temperature changes during electrophoresis. Higher voltage increases the temperature of the agarose gel which can denature DNA. This slight increase in temperature is maintained by the liquid medium of the buffer.
Buffer distributes charge evenly during electrophoresis as well.
Role of TBE/ TAE buffer in agarose gel electrophoresis
Tris is a strong base and borate is an acid, combination of both maintains the pH nearly neutral range of 8 to 8.5. Under this alkaline condition, DNA is protected and can separate properly.
EDTA has some important role to play in this combination. EDTA is a chelating agent. It chelates the Mg2+ ion which is required for enzyme DNAse as a cofactor. DNAse is an enzyme which cleaves DNA into small fragments.
So by addition of EDTA, our DNA is protected from the enzymatic activity. Further, the buffer will neutralize the charge of a water molecule.
Under the electrical current, the water molecule dissociates into H+ and OH-. The H+ ions can react with the PO3- of DNA. So the negative charge of buffer neutralizes the charge of the water and protects DNA.
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Though TAE buffer and TBE buffer works the same in electrophoresis, both have their own benefits and drawbacks.
TAE buffer can interfere with enzymatic reaction and protect DNA, in contrast, TBE buffer can not.
TAE has a lower buffering capacity while TBE has a higher buffering capacity as compared to TAE buffer. However, the borate can react with the sugar backbone of DNA, therefore, it is not always recommended.
Further, if glycerol is a component of your DNA gel loading dye, then TBE can be a major set back. The borate reacts with the glycerol and decreases the activity of DNA loading dye.
Hence if glycerol is the primary component in the DNA gel loading dye, TAE buffer is the best choice for getting a good result.
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Some of the important properties of TAE and TBE buffer are listed below:
- It has a lower buffering capacity so it can be exhausted by repeated use.
- The conductivity of TAE buffer is better so dsDNA can migrate faster as compared to TBE buffer
- DNA can be easily recovered in TAE buffer so the recovery rate of TAE buffer is higher.
- It is a cost-effective and cheaper than other buffer systems, further, the working solution requirement is lower as compared with TBE buffer (only 0.5X buffer is required).
- TBE has a higher buffering capacity due to the borate.
- Because of the lower conductivity, the migration of dsDNA is lower as compared with TAE buffer.
- The resolution is very good for longer DNA fragments.
- Borate inhibits many DNA enzymes hence the integrity of DNA is higher in TBE buffer.
- TBE buffer is a little costly and working solution requirement is higher as compared to TAE buffer (required 1X buffer).
- Borate from TBE buffer can interact with DNA so the recovery rate is lower as compared to TAE buffer.
- Conclusively, TAE buffer is most suitable and recommended than TBE buffer.
Preparation of TAE/ TBE buffer
During electrophoresis, a large volume of buffer is required. Generally, for 16 wells agarose gel electrophoresis unit, 1 to 1.2 litter buffer is required.
So always prepared a large quantity of buffer. Always prepare 10X stock of buffer. So we can Make 10 times 1X buffer during each run.
We have covered an article on how to prepare PCR primer based on our research knowledge, read the article here: PCR primer design guidelines
Preparation of 10X TBE buffer (for 250ml)
- Tris- 900mM
- Boric Acid- 890mM
- EDTA- 2mM
- Add distilled water to make the final volume of 250ml.
Add EDTA into Tris and boric acid and mix it until EDTA dissolve in water. Set pH nearly 8.0 to 8.5.
Now we have 250ml of 10X TBE buffer. Take 10ml from 10X TBE buffer and add 90ml distilled water which makes 100ml of 1X TBE buffer.
Similarly, for 1000ml buffer preparation, add 100ml of 10X TBE buffer in 900ml of D/W.
Preparation of 10X TAE buffer (for 250ml)
- 400mM Tris
- 200mM acetic acid
- 10mM EDTA
- Add D/W to make the final volume of 250ml
The preparation of 1X is similar to TBE buffer.
My ultimate guide for using electrophoresis buffer:
Freshly prepared reagents are key to get a good quality result. I always prefer to make 250ml 10X TAE buffer. This is enough for the entire day (If you are using electrophoresis 5 to 6 times a day).
If you prepare a 10X TAE buffer stock solution for 2 to 3 months, the buffering capacity will reduce as the salt is dissociated after some days.
Hence a clear DNA band will not be observed.
If you are performing a critical or sensitive experiment, Use buffer a single time in each run otherwise, for standardization and confirmation studies we can reuse the buffer.
If you have done electrophoresis so many times then you realize that after each run, some amount of buffer is dehydrated and the salt is dissociated onto both electrodes.
Read the article: PCR primer design guidelines
It shows that the buffering capacity of the running buffer is decreased and it is not used for another run of electrophoresis. So discard this buffer and prepare another batch of a fresh 1X buffer.
Always weigh each chemical properly. Never take weighing as granted because accurate weighing makes you a better researcher.
Use the same buffer for filling the tank and preparation of gel. Never use two different types of buffer for both (two different types of the buffer which mean buffer from two different batches of stock). This will alter the quality of the result.
Read the article: Role of MgCl2 in PCR reaction