Abstract:  Four types of DNA stains were evaluated based on safety, ease of use, cost, and resolution.  Ethidium Bromide was tested primarily as a comparison to the standard stain normally used in education, industry, and research.  While price is higher with the safer safe stains both FastBlast from Bio-Rad and Sybr Safe from Molecular Probes were found to have good resolution and be almost as easy to use as Ethidium Bromide without the toxicity and mutagen dangers.

Introduction: 
A large number of accidents occur every year involving chemical release in secondary schools.  In 15 states reporting in the HSEES, a department of the federal Agency for Toxic Substances and Disease Registry, 423 incidents were reported in the years 2002-2007, 31% of those resulting in injury.  While the largest number of these were mercury release which has a very low reported rate of injury (2%) the release of other chemicals such as Hydrochloric acid had a high rate of injury associated with the incident (58%.) ¹ 

Even though ethidium bromide is cheap, has good resolution and is relatively easy to use, it can be extremely dangerous in the hands of someone that is not ready to accept the responsibility to handle such a dangerous chemical.  It can easily be absorbed through the skin, eyes, or respiratory tract where it will bind tightly to DNA, acting as a mutinagen.  Spills in a lab are also very costly to clean up as well as disposal of ethidium bromide waste.²

There are currently in excess of 14 commercially available dyes available for staining that are considered safe.  Many of these have not been tested as well as ethidium bromide however.  Nile blue sulfate has been suggested as being safe and having high resolution as well. ³  SybrSafe as well as other Sybr stains have good visibility as well as being fluorescent and therefore more interesting for young students⁴.  They also have been proven to have a very low chance of having mutanagenic properties.⁵  The learning curve and a possible refinement of technique may be needed however for these dyes to possibly work, and to be easy enough to keep the attention of secondary school students.

These younger students tend to have a shorted attention span and trouble focusing on their technique compared to older students and researchers, as would be expected.  Many of the stains require distaining that can be time consuming, or difficult.  Schools also tend to have limited budget on which to buy supplies for their laboratories, making cost important as well.  Still, as colleges expect more and more to be learned prior to graduation, and in an attempt to get students excited about science and possible career and education paths, learning about DNA sciences could be of great benefit.

Though some information is becoming available on methods of safer protocols for secondary education⁶, we still have farther to go to making this information more accessible to high school and middle school science teachers.

Methods:   Buffer- TBE buffer was prepared and used in making all gels, loading buffer, and used as a running buffer.  A 10x buffer was made and diluted at time of use.

First a .3M EDTA stock was made by putting 111.66g of EDTA in 600 ml of distilled water, places on a stirrer and brought to a ph of 8 with Sodium Hydroxide and brought to 1 liter with more distilled water.  This was allowed to stir overnight to dissolve.  

A 10X stock of TBE buffer was then made using 54g of trizma base and 12g of boric acid added to ~800ml of Distilled water and placed on a stir plate.  To this was added 33.3ml of the .3M EDTA solution and volume was brought up to 1 liter.  Final concentrations are 45mM Tris-borate and 1mM EDTA.  Both may be placed in containers and stored at room temperature. 
As long as no precipitate forms they should be fine.

Marker and loading
Buffer- Lambda DNA hind III digest (Sigma-Aldrich) was used as a ladder for comparing resolution and visibility of DNA staining methods, and was loaded at 2ug, 1ug, and .1ug per well  for 3 lanes on all gels for comparison of resolution.

Additional loading buffer was used to get these concentrations.  The loading buffer was 60% glycerol, .3% (byweight) Bromophenol Blue and 37% distilled water.

Gels- Gels were all agrose type 1 (Sigma) at 1% concentration with running buffer. Apx 30-50 ml

total molten agrose per gel.

Gels were run at a voltage of 5v per cm space between electrodes.  In the case of the available

electrophoresis unit, 75 volts were used.

Stains-  Stains that were compared were Ethidium Bromide (for control), SybrSafe (Invotrogen), Fast Blast(Molecular Probes), and Nile Blue Sulfate.

Two methods were attempted for each stain; staining after electrophoresis, and precasting and running electrophoresis with stain in the running buffer (except for Ethidium.)

These were either visualized with a UV light box for Ethidium and Sybrsafe, or de-stained and viewed with a light box, or on a light background for the other two stains.

Staining after Electrophoresis-

Ethidium - 2μg/ml of running buffer was used to stain for 30 minutes agitating on occasion.

SybrSafe – Sybrsafe was diluted from 10,000X to 1x solution or 5μ for 50 ml of stain.  It was found that allowing the concentrate to come to room temperature before measuring greatly improved ease of use.  Gel was incubated for 30 minutes in a dark place, agitating occasionally.  A shaker table could also be used.⁷

Fast Blast – a 5:1 mixture with water was made and gel was soaked for 2-3 minutes.  Remainder of stain may be stored in a bottle and used for a total of 8 times before disposal.  Gel was then rinsed with warm tap water, changing tap water as it darkens as agitating for 30 minutes to overnight.⁸

Nile Blue Sulfate- 2μg/ml mixture with water, stained for 1-3 minutes.  Destain with warm tap water and agitation, changing tap water occasionally for 30 minutes to overnight.  Lower concentrations seem to work better as did overnight destaining.⁹

Pre-casting stains and running buffers-

Ethidium Bromide- .5μ/ml is added to molten Agrose and is cast into the gel. Load and run as normal.
SybrSafe – Dilute 1:10,000 into molten agrose.  Also use in running buffer at 1:10,000. ⁷

Fast Blast- Use 33μl per 50 ml agrose. As well as 200μl Fast Blast /300 ml buffer.  If necessary
destain after.⁸

Nile Blue Sulfate- Add 1μg/ml molten agrose and .25μg/ml in running buffer.  Destaining may be nessasary.⁹

Discussion: 
Ethidium Bromide was used as a control as it is the industry standard.  All comparisons were made to Ethidium.

Sybrsafe is Fluorescent like Ethidium, but it has a higher resolution.  As long as you allow the 10,000x concentration to warm a bit after removing it from refrigeration it is quite easy to handle.  Since most of this would be done by a teacher in secondary schools, making of the stock stain may not be as much of an issue.  For those that would have trouble, a dilute stock is available in a liter, but shipping cost is higher.  While cost is much higher than Ethidium and is the highest of the methods we compared, it is still not out of reach for most science programs at 55 cents per gel.  Its ease of use and the flashy showing of being a fluorescent stain make it a great stain in addition to it’s very high resolution.  Even smaller concentrations of DNA can be seen with this dye then with Ethidium.  With pre-casting it appears to have a strong affinity for the loading buffer that was used in this experiment.  If pre-casting is desired, a different loading buffer should be tried.

Fast Blast offers a great versatility as it can be used as a 5x concentration that may be used up to 8 times before disgaurding and distained or stained overnight at 1x concentration and visualized without distaining.  Destaining did take several rinses in warm water and worked even better destaining overnight.  Gels may be dried and stapled into lab books if so desired, though proper drying requires a gel dryer.  It’s resolution was comparable to that of ethidium bromide and it’s cost was 22 cents per gel.  If destaining is not seen as a problem, this may be the easiest of non-fluorescent stains. 

Since a UV light box is not required, this stain has some definite pluses.

Nile Blue Sulfate is cheap at 2 cents per gel, but was harder to work with.  If is does not completely dissolve it will leave dark spots on the gel that are hard to visualize through.  It was also difficult to destain, and visualization was difficult until overnight rinsing was used.  The resolution observed was a little less than that of ethidium.  If concentrations are lessened however, destaining may prove to be easier.  It is entirely possible that varying the concentrations of Nile Blue Sulfate in the staining, or precasting, that a better concentration may be found.  At the very low cost, this may be a worthwhile effort to try to optimize this process.

Stain Name	Price (per Gel)	Ease of use	Resolution	Safety
Ethidium Bromide	$0.05	Easy except for safety precautions	Very good	Mutanagen!
		Easy	Excellent	Low toxicity
Fast Blast	$0.22	Must destain (takes about 2 hrs)	Very good	Supposedly safe
Nile Blue Sulfate	$0.02	Must destain (takes about 5+hrs)	Fair	Supposedly safe

References:
¹Hazardous Chemical Incidents in Schools --- United States; MMWR Weekly; CDC, November 7, 2008,57(44);1197-1200

² MIT Green Chemistry Case Study Substitution of Ethidium Bromide with SYBR Safe® March 2006

³Adkins S.; Burmeister M.; Analytical Biochemistry, Volume 240, Number 1, 15 August 1996 , pp. 17-23(7)
⁴Qing Huang, Et al.; Clinical Chemical Laboratory Medicine. Volume 43, Issue 8, Pages 841–842, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2005.141, 01/08/2005

⁵Victoria L. Singer, et al; Mutation Research/Genetic Toxicology and Environmental Mutagenesis

 Volume 439, Issue 1, 2 February 1999, Pages 37-47

⁶Britos, L, Et al;Biochemistry and Molecular Biology Education.Vol. 32, No 5, PP. 341-347, 2004

⁷Sybr Safe DNA Gel Stain info sheet; Invitrogen, June 2007; http://www.invitrogen.com /etc/medialib/en/filelibrary/pdf.Par.46177.File.dat/SYBERSafe_FAQs.pdf

⁸Fast Blast DNA Stain (500x) instruction manual; Bio-Rad; 4110153 rev A; 2000; http://www.bio-rad.com/LifeScience/pdf/Bulletin_4110153A.pdf

⁹Staining DNA 2; National Centre for Biotechnology Education, 2006; http://www.ncbe.reading.ac.uk/dna50/stainingtable.html