Electrophoresis Stains

Errday I'm stainin'

Viable choices

Ethidium Bromide

• Cost of product: essentially nothing
• Cost of disposal: £125 per 60 litres. Therefore minimum cost with 50ml gels is 125 / (60000 / 50) = £0.104 a gel
• Cost, total: 10p per gel, perhaps more realistically 15p-20p per per gel (since we'll want to put other stuff in the bins).
• Usage: flouresces under UV at 254 nm wavelength.
• Other considerations: possible mutagen, carcinogen, and teratogen. (This is from tests on cultured cell lines. There is no direct evidence implicating EtBr as a carcinogen in any animal.
• Why it's viable: We know it works. It's quite cheap. We have to organise professional hazardous waste disposal anyway.

SafeWhite

• Info: http://nbsbio.co.uk/product.asp?pID=6241&cID=71
• Cost of product: £40 for 1ml. 2ul per reaction, therefore 40 / (1000 / 2) = £0.08 a reaction
• Cost, total: 8p per reaction, assuming 4 reactions per gel = 32p per gel.
• Usage: Add to samples not gel. Fluorescence excitation maxima at approx 290-320nm, emitting at 515nm
• Other considerations: must be refrigerated (4C).
• Why it's viable: Safe. Relatively cheap. Can be purchased in bulk for a saving of 1p per sample.
• Our experience: Never visualised any bands, with lots of testing. Most probably bands are there, but are too weak to see. Would need to improve visualisation method to try it again.

SafeView

• Info: http://www.nbsbio.co.uk/product.asp?pID=2490&cID=71
• Cost of product: £40 for 1ml.
• Usage: add to gel as with EtBr. Fluorescence excitation maxima at approx 290-320nm, emitting at 515nm

GelGreen or GelRed

• Info: http://www.bioscience.co.uk/product~88488 (for example)
• GelGreen and GelRed cost roughly the same amount and have the same protocols.
• Cost of product: £81 for 0.5ml, 5ul per 50ml gel, so 81 / (500 / 5) = £0.81 per gel
• Cost, total: 81p per gel.
• Why it's viable: Expensive, but safe.

Webgreen

• We have a tube of something called 'webgreen'. Not sure what it is, but we haven't been able to visualise anything with it, so as with safewhite, could try again once we have a better visualisation method.

Non-viable options

Often, these are stains which are not added to the sample and are mutagenic, placing them in the unenviable position of being as difficult to use as EtBr, but more expensive. They may also be nonviable for a variety of other reasons.

SYBR Green

• Info: http://www.sigmaaldrich.com/catalog/product/sial/s9430?lang=en&region=GB
• Cost of product: £242 for 0.5ml, produces 5 litres of staining solution. Assume 100ml of solution per gel = 50 gels = £4.84 a gel.
• Cost, total: £4.84 per gel.
• Other considerations: must be frozen (-20C); must be used as a post-stain (20-40 minutes after electrophoresis has finished).
• Why it's not viable: post-stain which produces hazardous waste.

SYBR Green, re-use of staining solution

• Info: As above.
• Cost of product: As above, but staining solution can be refrigerated and re-used "three to four times". This reduces the cost by 4.
• Cost, total: £1.20 per gel.
• Other considerations: Re-using the staining solution seems like a pain. Even with re-use, this is expensive stuff. We would have to re-use each solution 40 times in order to get in the same league as the other stains (and that doesn't take disposal into account).
• Why it's not viable: post-stain which produces hazardous waste.

SYBR Green as part of a readymix

• Info: http://www.sigmaaldrich.com/catalog/product/sigma/s4438?lang=en&region=GB
• Cost of product: £96.60 for 100 reactions, so £0.96 per gel, BUT Taq readymix itself costs around £100 for 100 reactions, so it is essentially free.
• Cost, total: This is a RT-PCR stain, which means the PCR vessel containing the stain would need to be disposed of as hazardous waste (I think).
• Why it's not viable: We're not doing RT-PCR yet, and we can't use it for traditional PCR, really.

Methylene blue

• Info: About £4.95 for 100ml. See eg http://cgi.ebay.com.au/ws/eBayISAPI.dll?ViewItem&item=220367085167&ssPageName=ADME:L:OU:AU:1123#ht_1288wt_1085
• Cost of product: 1ml in 100ml of water for post-staining. So £0.0495 per gel. Or, 0.1ml in 100ml of water, for £0.00495 per gel
• Cost, total: 5p per gel, or 0.5p per gel.
• Other considerations: About 100 times less sensitive than EtBr. Post-stain, which is annoying (and time consuming).
• Why it's not viable: Chances are we won't see anything with it, because it's not sensitive enough.

SYBR Safe

• Info: https://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/DNA-RNA-Purification-Analysis/Nucleic-Acid-Gel-Electrophoresis/DNA-Stains/SYBR-Safe.html
• Cost of product: £44.50 for 400ul, 5ul per gel, so £0.55 per gel
• Total cost: 55p per gel.
• Why it's not viable: Invitrogen won't sell anything to us. (but we can retry with them)
• Our experience: We tested 5ul of sybrsafe that was donated to us, and found it to give much fainter bands than EtBr. See below. Lane 6 on the EtBr gel and lane 4 on the sybr safe gel is 2ul of the cell culture gDNA we use as a positive control. The difference is clear. The remaining numbered lanes on each gel are either ladder, PCR products from our extractions or our gDNA extractions. None of these were visible on the sybr safe gel.

EtBr gel

SYBR Safe gel

Acridine Orange

• Info: http://www.sigmaaldrich.com/catalog/product/sigma/a6014?lang=en&region=GB
• Interestingly enough, it glows orange-red when bound to single-stranded nucleic acids, and green when bound to double-strnaded nucleic acids.
• Why it's not viable: Mutagen (though it may be worth trying, for the double/single strand differentiation alone)

Propidium Iodide

• Info: http://www.sigmaaldrich.com/catalog/product/sigma/p4170?lang=en&region=GB
• Why it's not viable: apparently not commonly used as a stain for PCR visualisation. Also, it's a mutagen.

GelStar

• Info: http://www.lonza.com/products-services/bio-research/nucleic-acid-and-protein-electrophoresis/nucleic-acid-electrophoresis/nucleic-acid-gel-stains/gelstar-nucleic-acid-gel-stain-10000x.aspx?WT.srch=1&gclid=CPfSoq-QzLECFQRTfAodDCAAbQ
• Why it's not viable: Mutagen.

EvaGreen

• Info: http://www.bioscience.co.uk/product~88421
• Cost of product: £175 for 5ml, 2.5ul per sample. £0.0875 per sample
• Total cost: For four samples, £0.35
• Why it's not viable: qPCR only, which we're not doing yet.

Crystal (aka gentian) violet

• Cost of product: very cheap
• Other considerations: Post-stain, which is annoying.
• Why it's not viable: very unlikely to be able to visualise less than 100ng => we probably won't see anything.

Discussion

Here is a Web page with more information and roughly similar costings to mine: http://bitesizebio.com/articles/ethidium-bromide-the-alternatives/

Here is a Word doc with information on various dyes and disposal methods, along with indications of which ones are mutagenic: http://www.facilities.rochester.edu/cu/ElectrophoresisBufferSolutionsandGelDisposalGuidelines.doc

Three comments on all this:

1. Even though it's really cheap, I don't like SYBR Green in readymix. It's designed for RT-PCR, which we're not doing at the moment. If we don't use it for RT-PCR, then we will end up visualising a ladder using a different method from the one we use to visualise the samples, which sucks: we won't know if we screwed up the visualisation or if the reaction didn't work (this problem is what led us to ethidium bromide in the first place).
2. Even with disposal costs, ethidium bromide isn't that bad. It's less expensive than SafeWhite (for 2 or more reactions per gel), but, to be fair, that's still not a significant difference (10p or so). If we handle EtBr properly and dispose of it correctly, we should be fine.
3. For £40, we may as well check out SafeWhite. It's not particularly cheap, and we will still have to retain LabWaste for waste disposal, but we won't need them to dispose of gels, so we won't need them to come as frequently.