May 05, 2022
Preparing 10x TBE Electrophoresis buffer
- 1Mboalab, Beneficial Bio;
- 2University of Cambridge, Beneficial Bio
Protocol Citation: Nadine Mowoh, Jennifer Molloy 2022. Preparing 10x TBE Electrophoresis buffer. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkkok5l5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 28, 2022
Last Modified: July 12, 2022
Protocol Integer ID: 58849
Abstract
TBE and TAE buffers are often used in procedures involving nucleic acids, the most common being electrophoresis. Tris-acid solutions are effective buffers for slightly basic conditions, which keep DNA deprotonated and soluble in water.
EDTA is a chelator of divalent cations, particularly of magnesium (Mg2+). As these ions are necessary cofactors for many enzymes, including contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic degradation. But since Mg2+ is also a cofactor for many useful DNA-modifying enzymes such as restriction enzymes and DNA polymerases, its concentration in TBE or TAE buffers is generally kept low (typically at around 1 mM).
TBE buffer is used to prepare agarose gels and as running buffer in agarose gel electrophoresis to separate and identify and analyse nucleic acids.
SCOPE:
This protocol covers the steps involved in making 1L of 10x TBE buffer from Tris, Borate and EDTA powders
DEFINITIONS:
TBE: Tris Borate EDTA
Guidelines
This procedure can be performed by laboratory staff that have been trained and have theoretical and practical skills in good laboratory practices. It can also be performed by molecular biology students or students of related fields under the supervision of a laboratory staff.
Materials
Chemicals
- Tris
- Borate
- EDTA
Equipment/Materials
- Sensitive or Electronic weighing balance
- Measuring cylinder
- Distilled water
- Magnetic stirrer
- 500ml beaker
Preparing 10x TBE buffer solution (1 liter)
Preparing 10x TBE buffer solution (1 liter)
40m
40m
Use an electronic balance to weigh out 108 g Tris base (CAS# 77-86-1, free base) into a 1000 mL ml or higher volume beaker ( depending on what volume of beaker is available and should be big enough to contain the powders to be dissolved).
40m
Weigh9.3 g EDTA, disodium salt dihydrate (Cas# 6381-92-6, C10H14N2Na2O8· 2H2O, MW: 372.24) into a weighing boat and pour into the beaker.
Note
Note that you can also use 0.5 Molarity (M) EDTA solution at 8.3 . In this case skip to Step 4. Then add 40 mL EDTA solution to the dissolved salts.
Measure 100 mL distilled or deionised water using a measuring cylinder, pour into the 1000 mL beaker and stir for 00:01:00 either manually or using a magnetic stirrer and flea.
Note
If the TBE buffer will be used for RNA work, ensure that RNase-free water is used.
1m
Adjust volume to 1 L by adding 900 mL of distilled or deionised water.
Place the beaker on top of a magnetic stirrer and put a magnetic flea in the solution. Allow to stir until you have a clear solution, this may take up to 00:30:00
30m
Check the pH using a pH meter or Universal indicator strips and record the result for quality control. A properly prepared solution should be 8.3 and should not require adjustment.
Autoclave (if sterile solution is needed) and pour solution into clean, autoclaved and labelled glass bottles.
Note
10 x TBE buffer is stable for up to 6 months at room temperature. The solution should be discarded if it becomes cloudy.
Make 1 x TBE working solution
Make 1 x TBE working solution
This buffer can be diluted to 1x each time before use by diluting 100 mL of 10x TBE with 900 mL of distilled water to have 1 L of 1x TBE buffer.