Last week, in my article about the perils of exposing DNA to UV light during cloning procedures, I mentioned a couple of stains that offer an alternative to ethidium bromide for DNA visualisation.
I this article I compare all of the available DNA stains (I know of) that can be used in electrophoresis to clarify the options available to you.
The classic DNA stain. Ethidium bromide is a flat molecule that fits between adjacent base pairs (intercalates) in the DNA double helix. It has UV absorbance maxima at 300 and 360nm, and can also absorb energy from nucleotides excited at 260nm. The absorbed energy is emitted as orange/yellow light at 590nm. The fluoresence of EtBr is significantly higher when intercalated than it is in aqueous solution. Protocol: Can be used in the gel at or as a post-stain at a concentration of 0.5 mg/L. Detection: UV light Sensitivity: Can detect bands of 1-5ng Toxicity: Toxin, mutagen, tetratogen and carcinogen according to a variety of tests but effects on higher organisms have not been proven (see here) Price: 0.00075 GBP/100mL gel (based on a cost of 15 GBP/g EtBr) References: Karsten, U. and Wollenberger, A. Anal. Biochem. 77, 464-470, (1977) Methylene Blue
Methylene blue is member of the thiazin family of dyes that bind ionically to DNA and RNA. Since it’s interaction with DNA/RNA is weak, methylene blue is not a very sensitive stain, but has the advantage that is detectable in the visible range. Destaining in water may be required for maximum sensitivity. Protocol: Post strain only, in 0.025% (w/v) methylene blue in water. Detection: Visible light. Sensitivity: 40-100ng bands are reported to be detectable after de-staining. In my own experience, only bands of 500ng and over are reliably detectable. Toxicity: Non-mutagenic. Somewhat toxic if ingested. Price: 0.000015 GBP/100mL of staining solution (based on a cost of 0.6 GBP/g) References: Yung-Sharp, D. and Kumar, R. (1989) Technique 1 (3) 183-187.
Crystal violet intercalates into DNA in a similar manner to ethidium bromide but is apparently less mutagenic. It’s major advantage is that it is detectable in the visible range – so no need for UV exposure. Protocol: Use in gels at a concentration of around 1.2 mg/mL Detection: Visible light Sensitivity: 100-200ng bands are reported to be detectable, but according to DK’s comment here on Bitesize Bio, 2ug+ may be required for clearly visible bands. Toxicity: Mutagen (less so than ethidium bromide) Price: 0.03 GBP/100mL gel (based on a cost of 0.28 GBP/g) Reference: Rand, K.N. Elsevier Trends Journals Technical Tips, Online, T40022, 1996. SYBR Safe
SYBR safe is a commercial DNA stain manufactured by Invitrogen. It is marketed as being less harmful than ethidium bromide, but this is debatable. It’s major advantage is that it is as sensitive as ethidium bromide but does not require UV light for visualisation. Protocol: SYBR safe is used as an in-gel stain only. It is supplied in ready-made buffers so the working concentration is unknown. Detection: For visualising fragments required for downstream applications, the best (although more expensive) option is to use a blue light box as the wavelengths used do not cause DNA damage. UV-transilluminators can also be used, although specific filters may be required. Sensitivity: As sensitive as ethidium bromide – bands of 1-5ng should be detectable. Toxicity: Documented as less mutagenic that ethidium bromide, but it’s acute toxicity is higher. You can read the white paper on SYBR safe toxicity here. Price: 0.96 GBP/100mL gel (Based on a cost of 38.70 GBP / 400ul of 10,000X SYBR safe) Reference:Invitrogen’s SYBR safe product page.
Gel Red is a commercial DNA stain manufactured by Biotium. It is marketed as being the most safe, sensitive and robust nucleic acid gel stain- less mutagenic than ethidium bromide, but more stable in storage than SYBR safe. Like ethidium bromide, Gel Red is visualised using UV light. Protocol: Gel red can be used as post stain or in-gel stain. It is supplied in ready-made buffers so the working concentration is unknown. Detection: UV: excitation at 300nm, emission at 595nm – so conventional UV transilluminators are sufficient. Sensitivity: Bands of 0.25ng can be detected, according to my calculations from the data available in the product brochure. Toxicity: Less mutagenic than ethidium bromide. Read the report here. Price: 1.9GBP/100mL gel (Based on a cost of ?19/100ul of 10,000x gel red)
Reference: Biotium’s Gel Red product page
Are you using a stain that I have not mentioned? Leave a comment.
One of the most crucial steps in any cloning procedure is the preparation of the vector. Get it wrong and your chances of success will be drastically reduced. The overall aim for a good vector preparation is to obtain a fairly concentrated stock of undamaged, fully digested plasmid DNA that is free from contaminants. Missing […]
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