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How Do YOU Stain Your DNA?

Visualization of DNA in gels is one of the most common procedures a molecular biologist can perform.  In a good day, I can run at least 4 different DNA gels!  When I was trained, ethidium bromide (EtBr) was the only viable option to easily visualize small amounts of electrophoresed DNA.  However, safer and more sensitive dyes are now currently available on the market.  Here is a quick look at several different options with respect to cost, sensitivity, ease of use and safety.

Fluorescent Dyes

Fluorescent dyes are highly sensitive dyes that allow visualization of small amounts of DNA.  They require transilluminators for excitation of the fluorescent molecule and in some cases, filters for documentation.  Many fluorescent dyes are considered potential mutagens, and therefore care should be taken during use and special hazardous disposal procedures are required.

Ethidium bromide (EtBr).  As mentioned above, EtBr used to be the mainstay for staining DNA gels.  It is an inexpensive dye (cost per gel ~ $0.30), can detect as little as 1 ng of DNA, and can be used during or after electrophoresis with little to no destaining required.  EtBr is excited with UV light and transmits as a reddish orange color visible by the naked eye.  UV illumination can lead to nicking of DNA and inhibit downstream cloning applications.  Furthermore, EtBr has become rather infamously known as a potential mutagen/carcinogen due to its ability to intercalate between DNA base pairs.  EtBr is therefore considered hazardous waste, and appropriate disposal measures must be followed.

SYBR® family of dyes.  The SYBR® family of DNA dyes sold by Invitrogen are marketed as safe, highly sensitive, fluorescent alternatives to EtBr.  They are considerably more expensive than EtBr.  Three different dyes with varying properties are available:  SYBR®Gold,  SYBR®Green, and SYBR®Safe.  SYBR®Gold is the most sensitive dye and can detect as little as 25 pg of DNA.  SYBR®Gold can be excited by both UV light or blue light transmission, which prevents nicking of DNA. SYBR®Gold exhibits the highest sensitivity when used to stain gels after electrophoresis; however, in-gel electrophoresis can also be performed.  SYBR®Gold is the least expensive of the SYBR dyes, at a cost of approximately $2.70/gel.  SYBR®Green exhibits similar safety and excitation/emission properties as  SYBR®Gold, with a little less sensitivity (60 pg DNA).  SYBR®Green cannot be used in the gel during electrophoresis, but can also be used for quantitation of DNA by real-time PCR. SYBR®Gold and SYBR Green dyes are considered potential carcinogens, and hazardous waste disposal procedures must be followed.  SYBR®Safe exhibits the same sensitivity as EtBr, but is a non-hazardous, environmentally friendly dye.  Similar to EtBr,  SYBR®Safe can be used in the gel during electrophoresis, however, blue light transmission can be used for excitation.

A word of caution: traditional EtBr filters for photographing gels cannot be used with the SYBR® family of dyes, so you may need to purchase special filters.  A blue light transilluminator will be required if excitation with this wave length is desired.

Alternative dyes.  Several other companies sell fluorescent dyes with properties similar to either EtBr or SYBR® dyes.

  • Biotium, Inc. markets Gel Red and Gel Green.  Both dyes have been engineered to prevent the dye from passing through the membranes of living cells, thus generating a nonhazardous dye.  Gel Red has the same excitation and emission spectra as EtBr, while Gel Green is excited using blue light.  The dyes are $90-100 for 0.5mL of a 10,000x stock solution.
  • GelStar, sold by Lonza Bioscience, is a highly sensitive dye (detection limit 20 pg) for use during gel electrophoresis.  GelStar can be visualized using a traditional UV transilluminator or blue light.  Similar to EtBr, GelStar is a potential mutagen, and is therefore considered hazardous.  Cost is $161 for 250 ul of a 10,000x solution.
  • EZ Vision (Amresco) is a nonhazardous fluorescent dye that is used during gel electrophoresis.  EZ Vision has the added convenience of being incorporated into a DNA loading dye, thus preparation of gels with dyes is not necessary.  Cost is $113 for 5 mL of a 6x loading dye.

Non-fluorescent Dyes

Although much less sensitive than fluorescent dyes, non-fluorescent dyes are generally more affordable, require no special equipment for visualization and are often non-toxic.  They also don’t require UV illumination of the DNA, which may inhibit downstream cloning applications.  They are excellent stains for teaching labs.

Thiazin dyes.  Several dyes in the thiazin family are used for DNA staining.  These dyes bind ionically to DNA and therefore are not considered mutagens.  BioRad sells Fast Blast DNA, which can be used during or after electrophoresis.  The cost is $28 for 100 mL of a 500x solution.  Methylene blue powder can be purchased from many manufacturers and used to make a staining solution (0.002-0.02% in agarose gel running buffer).  Methylene blue can only be used post electrophoresis.

Nile blue sulfate.   Nile blue sulfate, or Nile blue A, is a cationic dye that can be used to visualize DNA during electrophoresis.  The dye is used both in the gel and in the gel buffer.  It is sold by several chemical suppliers ($32/Sigma).  At higher concentrations, Nile blue my change the migration of DNA and inhibit resolution.

What DNA stains do you like to use…and why?

 

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