Migration of Bromophenol Blue and Xylene Cyanol in Agarose Gel Running in TBE or TAE Electrophoresis Buffer

Bromophenol blue and Xylene cyanol are the two most commonly used tracking dyes for the analysis of DNA on agarose gel electrophoresis. Since DNA migration can not be seen during electrophoresis, these tracking dyes help to monitor the progress of agarose gel electrophoresis. Therefore, it is important to know the position of tracking dye and their corresponding migratory double standard DNA size fragments. This helps to determine how far a gel must be run without accidentally letting the DNA fragments of interest run out of the gel and at the same time ensuring good resolution among different size DNA fragments.

It is important to mention that the position of tracking dyes in relation to double standard DNA fragments is not stable. It is affected by gel percentage as well as different types of electrophoresis buffer used for gel run. The table below summarizes the position of DNA tracking dyes, bromophenol blue and xylene cyanol, in relation to the size of double standard DNA fragments in TAE and TBE electrophoresis buffers and agarose gel percentage.

Position of bromophenol blue and xylene cyanol in agarose gel in relation to the position of double standard DNA fragment in TAE (1x) and TBE (0.5 x) electrophoresis buffer.
Agarose gel, %Bromophenol blue  position corresponding to the double-standard DNA fragment, in base pairXylene cyanol FF position corresponding to the double-standard DNA fragment, in base pair
 TBE buffer (0.5x)TAE buffer (1x)TBE buffer (0.5x)TAE buffer (1x)
0.5 *∼750∼1150∼13000∼16700
0.6∼540∼850∼8820∼11600
0.7∼410∼660∼6400∼8500
0.8∼320∼530∼4830∼6500
0.9∼260∼440∼3770∼5140
1.0∼220∼370∼3030∼4160
1.2∼160∼275∼2070∼2890
1.5∼110190∼1300∼1840
2.0∼65∼120∼710∼1040
* For example in 0.5% agarose gel, Bromophenol blue migrates at approximately 750 bp long double standard DNA fragment in TBE buffer and at approximately 1150 bp long double standard DNA fragment in TAE buffer.

REFERENCES

  • Thermofisher: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012614_Gen_Recommend_DNA_Electrophoresis_UG.pdf

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