DNA Staining


DNA staining can be done with 2 types of dyes: Florescent dyes and non florescent dyes


Florescent 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

NAME

DESCRIPTION

PROS

CONS

Ethidium bromide (EtBr). 

 

    • EtBr is excited with UV light and transmits as a reddish orange color visible by the naked eye.
    • intercalates between DNA base pairs.
    • inexpensive dye
    • can detect as little as 1 ng of DNA
    • can be used during or after electrophoresis with little to no destaining required
    • UV illumination can lead to nicking of DNA and inhibit downstream cloning applications.
    • potential mutagen/carcinogen  

SYBR

Three different dyes with varying properties are available:  SYBR®Gold,  SYBR®Green, and SYBR®Safe.  

    • Safe
    • highly sensitive
    • fluorescent alternatives to EtBr.
    • more expensive than EtBr.

     

    1. SYBR Gold
    • most sensitive dye 
    • exhibits the highest sensitivity when used to stain gels after electrophoresis; however, in-gel electrophoresis can also be performed.
    • SYBR Gold dye can be used to stain double or single-stranded DNA​ or to stain RNA. 
    • It penetrates thick
    • an 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.
    • least expensive of the SYBR dyes
    • The dye exhibits 1000-fold greater UV fluorescence once it is bound to nucleic acids.
    • high percentage agarose gels and can be used in formaldehyde gels.​

    fluorescence of the unbound molecule is so low, destaining is not required

    •  potential carcinogens

     

    1. SYBR

    Green 

    • less sensitivity (60 pg DNA)

     

    •  used for quantitation of DNA by real-time PCR
    • cannot be used in the gel during electrophoresis
    • potential carcinogen

     

iii. SYBR Safe 

 

 

    • exhibits the same sensitivity as EtBr
    • suitable for staining RNA in gel

     

    • non-hazardous, environmentally friendly dye
    • used in the gel during electrophoresis, however, blue light transmission can be used for excitation.
    • supplied as either a concentrate or a ready-to-use solution 
    • special filters required.

Gel Red and Gel Green.

 

 

    • Gel Red has the same excitation and emission spectra as EtBr, while Gel Green is excited using blue light. 

     

    • engineered to prevent the dye from passing through the membranes of living cells
    • nonhazardous dye.
    • expensive

GelStar

 

    • highly sensitive dye (detection limit 20 pg) for use during gel electrophoresis.

     

     

    • an be visualized using a traditional UV transilluminator or blue light.

     

    • potential mutagen,

     

EZ Vision (Amresco)

 

    • incorporated into a DNA loading dye.
    • used during gel electrophoresis

     

    • preparation of gels with dyes is not necessary
    • nonhazardous
    • expensive

Eva Green

 

    • applications like quantitative real-time PCR.
    • good choice if you are using low-melting point gels for recovery of DNA.
    • very stable at high temperatures
    • highly fluorescent when bound to DNA.
    • low or no cytotoxicity or mutagenicity.

     

    • expensive

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.

 

 

    • dyes bind ionically to DNA and therefore are not considered mutagens.
    • Fast Blast DNA, which can be used during or after electrophoresis.
    • 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

     

    •  At higher concentrations, Nile blue my change the migration of DNA and inhibit resolution.

     





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