Staining Aragose Gels with Carolina BLU(TM) DNA Stain

Shirle Pace
Biotechnology Department

Students active in DNA science are eager to visualize the bands of DNA migrating through the agarose gels. Developed in cooperation with Dr. Greg Freyer of Columbia University, Carolina BLU(TM) stain has the ability to stain DNA during electrophoresis, allowing for early visualization of the bands. With its more intense staining and easier visualization of smaller bands of DNA, which are not usually seen after the destaining of methylene blue, now many gels are ready for visualization immediately after electrophoresis. This allows for faster analysis and interpretation of the results. This is accomplished by adding a small amount of a concentrated solution of the stain to the gel and buffer; the stain is picked up by the DNA as it migrates through the agarose gel. During electrophoresis, the positively charged stain particles in the agarose migrate towards the negative electrode of the gel box, thus the necessity for adding stain to the buffer. As the DNA passes the midpoint of the gel in its migration towards the anode, without the replacement stain from the buffer, it will actually lose stain in the clear portion of the gel. After electrophoresis, additional staining of the gel with a dilute solution of Carolina BLU(TM) stain in a staining tray intensifies all the bands with short staining (15-20 minutes) and short destaining (1 hour or less) times. Easy agitation of the gel in the tray facilitates staining and destaining. Distilled or de-ionized water should always be used for destaining because tap water contains chloride ions that remove stains from DNA and agarose within a few hours. Changing the water a few times speeds removal of the stain for easy visualization in less than an hour. Gels can be stored in a few milliliters of water in a covered stain tray for several weeks and even refrigerated in an airtight bag for a few months.

For optimum performance, consider the following tips. Less DNA is required in a digest of lambda DNA than if you were using methylene blue. Specific amounts of lambda and plasmid DNA are listed in the package instructions. Because temperatures above 50degrees C cause the stain to fade to ineffectiveness, melted agarose is best cooled until the container is comfortable to the touch before adding the appropriate microliters of Carolina BLU(TM) stain. Follow the instructions exactly for adding stain to the gel and buffer because extra stain can cause artificially precipitated DNA bands in the gel, which detracts from the usual banding pattern. Strictly following stain amounts for your voltage requirements provides excellent results in gels, which can be stored for future viewing and analysis. The final stain can be reused eight times, and buffers can be reused four or five times unless stain was added. Buffer with stain added can be reused one time in the same day, but it fades and loses its effectiveness if stored longer. Gels and buffer may be prepared one day early for lab the next day. Gels may be photographed with the same cameras, white lights, and yellow filters used for methylene blue photography. Safe and easy cleanups are another advantage of Carolina BLU(TM) stain.