Physical property-Stability and pH (TBE and Borax Agarose electrophoresis buffer)
Stephane Fadanka, Nadine Mowoh, Mujar Minette Shalo
Abstract
The pH check is important because changes in the pH might affect mobility of DNA in the agarose gel hence PCR result alteration and misinterpretation.
The physical stability of the electrophoresis buffer is important because if the powder absorbs moisture from air overtime, they might not be very efficient in allowing migration of DNA samples in the gel which could affect the PCR results.
This protocol describes how to assess the physical property of TBE and Borax electrophoresis buffers.
Before start
Ensure all preliminary steps involved in the process are performed (e.g initially weighing and drying buffer powders)
Steps
Confirming Stability and pH
To confirm the weight of the TBE powder buffer sachet:
- Weigh the individual buffer components that would make 500 mL of the 10x buffer as indicated in the table below ( the individual powders could be pre-dried if necessary in an incubator with silica gel beads at
37°C).
| A | B |
|---|---|
| Component | Amount in grams (g) |
| Borax | 27.515 |
| Tris | 53.905 |
| EDTA | 3.72 |
Table 1
- Put them together in a beaker and mix.
- Pour the powder mix in a zip lock sachet and store in an air tight container for 1 to 3 days.
- After, open the sachets and pour the powder into a weighing boat and measure the weight of the dried powder.
- The weight should be approximately close to or equal to the weight of the powder before the 3 days storage.
To confirm the pH of the buffer solution:
- Pour the powder buffer into an appropriate size beaker and add the appropriate amount of distilled water as indicated on the powder sachet (e.g 1 sachet in 500 mL distilled water).
- Mix until a clear solution is obtained
- Use a pH meter to check the pH of the solution which should be between 8.3 and 8.5.
