Team:Glasgow/AzureA

Glasglow

Azure A Staining

Summary

Figure 1: The Glasgow 2015 iGEM Azure A staining equipment

Azure A (Dimethylthionine) is a blue dye of the Thiazin family which can be used to visually stain DNA down to quantities of 20ng. Azure A staining requires no expensive equipment or controlled disposal techniques like the common DNA stain Ethidium Bromide. Glasgow’s 2015 iGEM team utilised Azure A staining for the vast majority of the Agarose gel DNA purification/extractions we performed. We aim to promote Azure A staining in order to expand the participation of community and high school labs in iGEM and molecular biology, where they would not be able to meet the costs of equipment or disposal of Ethidium bromide for DNA visualisation.

Background

Azure A is produced when Methylene blue, another Thiazin stain, is oxidised. Methylene blue is less sensitive for staining DNA than its oxidation products, while Azure A demonstrates reduced background gel staining and a reduced required staining time (NCBE, 2003b). The Thiazin family is thought to stain nucleic acids through ionic interactions with the phosphate groups of the sugar-phosphate backbone (NCBE, 2003a); but weak intercalative interactions with DNA have also been described (Paul and Kumar, 2013).

Figure 2: Chemical structures of four of the Thiazin family of dyes, including Azure A and Methylene blue.

Thiazin dyes are combined with Eosin to make Giemsa stain, a ubiquitous diagnostic stain for intracellular protozoan parasites such as Malaria and Trichomonas. Giemsa stain is also utilised to visualise chromosomal configurations by the so-called “G-Banding” of karyograms, which were the early methods of detecting chromosomal deletions and translocations (Sumner, 1980).
The Azure A compound used herein was Azure A chloride; supplied as a dark green powdered solid available from several chemical supply vendors. The Azure A used by Glasgow Team was purchased from Sigma-Aldrich (A6270)

The costs of staining DNA

Originally, the system containing UirS, UirR, and PlsiR accounts for a negative phototactic response to unidirectional UV-A light. The proposed mechanism puts UirS, a transmembrane protein of the CBCR family, as the molecule that perceives UV light. It is suggested that through a physical interaction between UirS and UirR and possibly a phosphotransfer from UirS to UirR, UirR is released from the transmembrane protein. The released UirR can now bind to DNA and UirR, which is similar to other activators of stress responses, was found to be a transcriptional activator of lsiR after binding to its promoter PlsiR .

Sigma-Aldrich sells 10ml of Ethidium bromide (EtBr) solution at a concentration of 10mg/ml for £45/$56 (E1510) (Sigma-Aldrich, 2015). We used a solution of 15ul (10mg/ml) EtBr per 0.5L of TAE running buffer for post-run staining, which works out to ~6666 0.5L stains per bottle of Ethidium. Traditional DNA staining with Ethidium bromide appears to be low-cost when only the cost of the stain is considered; with a cost per gel of £0.0067. However the cost to a lab is greater than simply the stock stain; Ethidium stained DNA requires visualisation on a UV-transilluminator, and the model of UV transilluminator available to us comes from a range which begins at £600/$900 (VWR, 2015). UV illumination requires an enclosed space where other people and sensitive items will not be damaged by irradiation. Additionally, imaging an Ethidium stained gel requires specialist camera filters to prevent camera sensor damage by the UV radiation, or the use of an enclosed illumination and photographing apparatus such as the BioRad Gel Doc™ XR system. Disposal of Ethidium bromide stained gels is also costly; EtBr has been found to be a potent mutagen in in vitro testing, thus disposal and handling is treated very seriously. The EtBr disposal policy of the University of Glasgow states that EtBr waste should be disposed of into biohazard marked containers, which must be uplifted by a 3rd party waste disposal service.

10g of Azure A chloride powder costs ~£36/$60. 500ml of 1x stain was reused by Glasgow up to 2x per day, for two weeks, equalling somewhere in the region of 28 gels per 500ml of 1x. 10g of Azure A chloride will produce 25L of 1x stain, or 50 500ml batches. This calculates to an estimated 1,400 gels per 10g of Azure A; or £0.026/$0.042 per gel. At face value this is more expensive per gel than EtBr, but Azure A does not require specialist disposal services, the lightbox which is helpful for visualising bands for gel extraction can vary in price but will not likely reach the price of a UV transilluminator, with a Top of the Range professional white light box for photography purposes costing ~£282/$440 (Calumet: https://www.calphoto.co.uk/product/Calumet-Light-Box-A3-/710-407X) with cheaper alternatives available. Imaging an Azure A gel can be done on any lab or office scanner, if the gel is placed between two sheets of acetate, rather than requiring specialist UV illumination and capture equipment.

Figure 3: Sequential destains of one Azure A stained 1% Agarose gel, following the protocol provided below, imaged between two sheets of Acetate on a office scanner. Click for full size.

Protocol for producing a 2x stock solution of Azure A stain

We maintained a 2x stock solution in line with the recommendation of the NCBE guidance from which we first learned of Azure A staining (NCBE, 2003b).
To produce 1L of 2x Azure A (0.08% Azure A/40% Ethanol):
• Dissolve 0.8g Azure A chloride solid in 1L 40% Ethanol
• Store in a glass screw capped bottle in darkness, or alternatively wrap the bottle in aluminium foil. Azure A will bleach over time in sunlight and lose effectiveness.
• Prior to use, a 1x (0.04% Azure A/20% Ethanol) solution should be prepared by diluting the 2x stock 1:1 with distilled or deionised water to a desired final volume.
Approximately 500ml of 1x stain was found to be suitable for staining of gels used in our experiments, however this may vary depending on gel and staining tray size.
Similarly 1x should be stored in a glass screw capped bottle in darkness.

Protocol for staining with 1x Azure A

Azure A is used as a post-run gel stain. The effect of putting it in the running buffer is unknown, other than it will definitely stain the gel-kit blue.
• Transfer the gel to a tray or container.

• Glasgow team used a large clear plastic Tupperware box with a lid. A lidded container is preferred to prevent accidental overflowing of the stain over the side.

• Azure A can stain plastics so any tray used for Azure A should not be utilised for other uses unless it has been thoroughly cleaned with 100% Ethanol.

• Pour enough 1x Azure A solution into the container to cover the gel, without overfilling it.

• Shake the container for 10-15 minutes, either by benchtop shaker on a low setting or by hand (fun)

• The 1x stain was shown to be useable for a couple of weeks before making up a new batch, without any demonstrable contamination issues or reduction in effectiveness.

• After a 10-15min stain, strong DNA bands may already be visible at this stage, but we recommend at least one round of destaining for improved visualisation.

• To destain, cover the gel in the used running buffer from the gel-kit, or distilled/deionised water. Shake for 10-15 mins. The destaining solution should be discarded into a sink drain along with running water.

• Repeated rounds of destaining will improve visualisation of weak bands.

• Band visualisation is also improved by viewing the gel on a white lightbox, either inside a clear plastic staining container, or on top of a sheet of acetate to prevent staining of the light box.

Gel extraction using Azure A

Gel extraction after staining with Azure A follows the same protocol as an Ethidium bromide gel extraction, without the need to use a UV transilluminator and associated face protection.

As the Glasgow team used a clear plastic Tupperware box to stain our gels, we often proceeded to place them straight on top of our benchtop lightbox after destaining and perform the gel extraction there, without having to manipulate the gel onto another surface.

Figure 4: A typical Azure A gel following extractions with a scalpel

The scalpel blade should be cleaned between sequential fragment extractions from the gel to prevent contamination.

DNA extracted from gels following electrophoresis was purified using a QIAquick Gel Extraction Kit by QIAGEN. The only difference caused by use of Azure A was a change of colour not normally seen in use of the kit; after addition of yellow Buffer QG for the fragment melting stage the colour changes to green, due to the interaction of the blue and yellow dyes in the gel fragment and QG respectively.

N.B. This is not the same erroneous colour change that is warned about in the protocol due to issues with pH.

Figure 6: The inconsequential colour change observed after addition of Buffer QG from the QIAquick Gel Extraction Kit.

Comparison of Azure A staining to Ethidium Bromide staining

We attempted to demonstrate the DNA staining sensitivity of Azure A and Ethidium bromide through a repeatable, directly comparable experimental protocol.

iGEM transformation efficiency control DNA (J04450 (RFP) in pSB1C3) was transformed into chemically competent DH5a cells, and then a resulting transformant colony was inoculated into a 10ml overnight culture. Plasmid DNA was isolated from 4.5ml of an overnight culture following the standard QIAGEN “QIAprep Spin Miniprep Kit” protocol.From the resulting J04450.pSB1C3 miniprep a 1/100 dilution was created and OD260 was measured in a spectrophotometer to determine the DNA concentration. DNA concentration of the miniprep DNA was calculated to be ~285ng/ul from the spectrophotometer reading.

Restriction digests were set up with EcoRI-HF enzyme and an “uncut”control Serial dilutions of the cut and uncut DNAwere made in order to get a range of known DNA concentrations to load onto two gels;

1. Stained with Ethidium bromide and imaged in a Bio-rad Gel Doc XR imager,
2. Stained with Azure A (destained repeatedly over 2 hours) and imaged on a standard office scanner between two sheets of acetate.

The results (Fig 7 & 8 below) show that Ethidium bromide could resolve bands of DNA as low as 2ng, whereas with Azure A band with ~17ng of DNA was the lowest resolvable on an office scanner after 3 hours of destaining.

Figures 7 & 8: 1% Agarose gels loaded with an identical dilution series of prepared DNA of known concentration; one gel stained with Azure A and one gel stained with Ethidium bromide (For EtBr staining method see: Protocols)

Conclusions

Azure A is good if you are using relatively high (or well-known) amounts of DNA ? Glasgow iGEM team believe that the safety improvements far outweigh the limited sensitivity and would recommend using Azure A staining, especially where stringent safety procedures and environments required for EtBr staining cannot be met.

Safety and considerations of Azure A

Azure A’s Material Safety Data Sheet (MSDS) is available from its supplier; it is not deemed to be hazardous.
Azure A solution can be disposed of down a conventional sink.
Azure A stained Agarose gels can be disposed of in standard laboratory waste.
Standard laboratory safety measures for handling a dye; such as gloves, goggles, lab coat, should be followed when using Azure A.
Flames should not be used near to preparation of Azure A solution due to flammability.
Azure A solutions will stain clothing and surfaces if spilled.
Azure A solution can be removed from surfaces (such as a benchtop or staining tray) with 100% Ethanol and paper towel.

NCBE. 2003a. National Centre for Biotechnology Education | DNA50 | Staining DNA [Online]. Available
NCBE. 2003b. National Centre for Biotechnology Education | DNA50 | Staining DNA Table [Online]. Available
PAUL, P. & KUMAR, G. S. 2013. Spectroscopic studies on the binding interaction of phenothiazinium dyes toluidine blue O, azure A and azure B to DNA. Spectrochimica Acta Part a-Molecular and Biomolecular Spectroscopy, 107, 303-310.
SIGMA-ALDRICH. 2015. Ethidium bromide solution BioReagent, for molecular biology, 10 mg/mL in H2O | Sigma-Aldrich [Online].
SUMNER, A. T. 1980. DYE BINDING MECHANISMS IN G-BANDING OF CHROMOSOMES. Journal of Microscopy-Oxford, 119, 397-406.
VWR. 2015. UV transilluminators, Benchtop series [Online]. Available