Cresol red is a triarylmethane dye and it can be used as an alternative loading dye for gel electrophoresis to monitor the progress of a running gel.
This dye is also used as an acid-base indicator, or pH indicator. pH indicators give an approximate value of pH of a solution. As a pH indicator, when cresol red is added into a solution, a color of the solution can then be observed.
For example, when you add cresol red into an acidic solution, you’ll see that the dominant color is yellow. After you add base into the solution, it will slowly turn color. As you keep adding base into the solution, there will be enough of the red form of the cresol red, so the solution will start to change from yellow to red. Eventually, all yellow in solution turns red as more base is added. In fact, this color change indicates your solution now has become more alkaline (basic).
What are cresol red used for?
Cresol red as a pH indicator
One of the primary uses for cresol red is in a histochemical method. For example, researchers used cresol red to find out about the pH of the digestive tract of mosquitoes (del Corena et al., 2005). In the study, the mosquitoes were fed on meals containing cresol red, and then the researchers observed the color change of the mosquito’s guts. The researchers then compared the color of the guts with the color of the cresol red solutions that had different pH.
Cresol red as an electrophoresis indicator
In addition to a pH indicator, cresol red can also be used as a molecular weight marker in the agarose gel electrophoresis and polyacrylamide gel electrophoresis to track the progress, so your DNA, RNA, or protein will not run off the gels.
When a loading dye containing cresol red is run on the gel, a colored band would appear and run at the same rate as a 125 basepair (bp) DNA molecule. However, it depends on the percentage of the gels, the type of buffers, and other factors.
If used in the loading dye, the gels have much less shadow, particularly for ethidium bromide stained gels, so you can see your DNA bands clearer. What we mean by shadow is some dyes can cause darker zones on your gel, which can hinder visualization.
Cresol red and PCR time-savings
To save time and prevent cross contamination between samples, you can add cresol red in the PCR master mix before performing your PCR. Typically, the addition of cresol red will not interfere with Taq Polymerase. For example, in a 20 µl of reaction, you can add 4 µl of 5x Cresol Red/sucrose loading buffer.
Cresol red as an amplification indicator in isothermal reactions
Another use for cresol red has to do with isothermal amplification. If you’re not familiar with isothermal amplification, we have a really detailed article on what it is and the different types of methods out there. But essentially, isothermal amplification is continuous nucleic acid amplification without cycled temperature changes.
In this case, cresol red is used specifically for an isothermal amplification technique called isothermal multiple-self-matching-initiated amplification (IMSA).
In a study, researchers incorporated cresol red to detect the presence of a virus, porcine circovirus (PCV3) (Gou et al., 2020). The researchers observed the reaction containing PCV3 capsid gene DNA changed color from red to yellow after incubation at 63°C for 60 minutes when cresol red was used. They also observed the negative control maintained the red color. Therefore, researchers used cresol red as an indicator of amplification by observing the color change.
What is the pH range for cresol red?
For cresol red, the transition pH in which the color starts to change is at pH 7.2–8.8.Cresol red is yellow below pH 7.2 and red at pH 8.8 and above. The change of color from yellow to red doesn’t occur sharply at a certain pH, but instead there is a gradual change of color at a particular pH range.
How to make cresol red indicator solution
- Mix 2.65 ml of 0.1M sodium hydroxide and 20 ml of 95% ethanol solution.
- Add 0.1 g of cresol red in the solution
- Add distilled water to make 100 ml of total solution.
How to use cresol red to make 5X direct-loading PCR buffer
5x Direct-loading PCR buffer (Carlson, n.d.)
- Dissolve 3 g of sucrose in 5 ml of distilled water.
- Add 150 µl 1M MgCl2.6H2O.
- Add 500 µl 1M KCl.
- Add 400 µl 1M (NH4)2SO4.
- Add 500 µl 1M Tris-Cl, pH 9.0.
- Add 0.003 g cresol red.
- Add 250 µl 10% IGEPAL CA-630.
- Mix well and centrifuge briefly.
- Add distilled water to make 10 of total solution
- Sterilize with 0.22 µl filter
- Aliquots and store at -20̊C.
- Use 4 µl in 20 µl of PCR reaction.
Find GoldBio products below:
Cresol Red (Catalog No. C-830-10)
Agarose LE (Molecular Biology Grade) (Catalog No. A-201)
High Resolution Agarose (For Nucleotides < 1kb) (Catalog No. A-202)
Low Melt Agarose (Catalog No. A-204)
1 kb DNA Ladder (Catalog No. D010)
1 kb PLUS™ DNA Ladder (Catalog No. D011)
VersaLadder™, 100-10,000 bp (Catalog No. D012)
Carlson, A. (n.d.). Schwer Lab 5´ Direct-loading PCR buffer. Retrieved May 19, 2021, from https://schwerlab.ucsf.edu/sites/g/files/tkssra1001/f/wysiwyg/Protocols/Schwer_DirectLoading_PCR_Buffer_0.pdf.
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del Pilar Corena, M., VanEkeris, L., Salazar, Ma. I., Bowers, D., Fiedler, M. M., Silverman, D., Tu, C., & Linser, P. J. (2005). Carbonic anhydrase in the adult mosquito midgut. Journal of Experimental Biology, 208(17), 3263–3273. https://doi.org/10.1242/jeb.01739.
Finnerty, J. (n.d.). Agarose Gel Electrophoresis The Principal. http://people.bu.edu/jrf3/BI550/BI550_Marine_Genomics/Molecular_Biology_Protocols_&_Notebook_Sheets_files/BI550_2013_LabTechnique_Electrophoresis.pdf.
Gou, H., Bian, Z., Cai, R., Jiang, Z., Song, S., Li, Y., Chu, P., Yang, D., Zang, Y.-A., & Li, C. (2020). The Colorimetric Isothermal Multiple-Self-Matching-Initiated Amplification Using Cresol Red for Rapid and Sensitive Detection of Porcine Circovirus 3. Frontiers in Veterinary Science, 7. https://doi.org/10.3389/fvets.2020.00407.
pH indicator solutions. (2021). Arizona.edu. http://www.atmo.arizona.edu/students/courselinks/fall12/atmo170a1s1/lecture_notes/SO2/pH%20indicator%20solutions.html.
Preparation of Indicator Solutions: Pharmaceutical Guidelines. (n.d.). www.pharmaguideline.com. Retrieved May 14, 2021, from https://www.pharmaguideline.com/2010/09/preparation-of-indicator-solutions.html.
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