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| '''The following information is for basic lab technique.''' | | '''The following information is for basic lab technique.''' |
| *Also see [[DIYbio:Notebook/Safety_Manual_1.0 | the DIYbio Safety Manual]]. | | *Also see [[DIYbio:Notebook/Safety_Manual_1.0 | the DIYbio Safety Manual]]. |
| + | |
| + | == Safely Staining DNA == |
| + | |
| + | "My personal foci are safety, ease of |
| + | visualisation (non-UV please), impact on DNA (no UV please!!) and ease of |
| + | disposal. |
| + | *Methylene blue* has been discussed before. Apparently using a lower |
| + | concentration can reduce background staining while not affecting sensitivity |
| + | to DNA (that's a matter of DNA quantity, not methylene blue concentration). |
| + | Unfortunately it takes between 3 and 5 times as much DNA to absorb |
| + | noticeable amounts of methylene blue, compared to EtBr. Also, it degrades |
| + | over time, so keep in the fridge, minimise contact with oxygen and cover |
| + | container with tinfoil. |
| + | *Crystal Violet* can apparently be used, and is moderately safe. It's |
| + | generally considered safe as an alternative antifungal treatment for |
| + | breastfeeding mothers (my sister has used it lots), so I think it qualifies |
| + | as a "home safe" dye. There are some very mild carcinogenic concerns, but |
| + | they apply to regular consumption rather than the occasional topical |
| + | exposure. |
| + | The method detailed in a closed-access paper I've skimmed goes roughly like |
| + | this: Run DNA in an unstained Gel, then stain the gel for 30 mins with |
| + | 0.001% Crystal violet. Apparently this is more sensitive than EtBr. For |
| + | better sensitivity, use 0.0025 CV with 0.0005% *Methyl Orange*, if you can |
| + | get some. Visualise on a regular light box, not UV. Paper: "Counterion-dye |
| + | staining method for DNAin agarose gels using crystal violet and methyl |
| + | orange." |
| + | According to this: |
| + | http://www.ncbe.reading.ac.uk/ncbe/PROTOCOLS/DNA/PDF/DNA14.pdf |
| + | You can also use "*Nile Blue*" as a dye, and this is used in some/many |
| + | commercial running buffers that let you see the DNA moving during |
| + | electrophoresis. This stuff isn't perfect, see the MSDS: |
| + | http://www.sciencelab.com/msds.php?msdsId=9926230. It's also not exactly |
| + | commonly available, being used as a histology stain or a photosensitiser in |
| + | light-directed chemotherapy. Worth noting though. |
| + | I've seen several alternatives to Sybr-Safe that are chemically a mystery, |
| + | but you can find some for sale here: |
| + | http://nbsbio.co.uk/categories.asp?cID=75. According to the sales rep I was |
| + | talking to, the *Safeview/Safewhite* stains are blue-light excitable, even |
| + | if the datasheet disagrees. I suspect "*Visual violet*" is a formulation of |
| + | Crystal Violet, used as an in-gel stain. They also have "*EZ-Vision*", for |
| + | which more detail is available here: |
| + | http://www.mobitec.de/de/products/bio/02_genomics/ez-vision.html. Apparently |
| + | it requires transillumination. |
| + | So, there's a bunch of alternatives. My money's on Crystal Violet and |
| + | Methylene blue. You might even be able to combine the two for the |
| + | best-of-both approach: Meth blue seems to bind the outside of DNA helix, so |
| + | if CV intercalates into the actual base-pairing site there might be no |
| + | conflict." |
| + | :: -- Cathal Garvey, Aug 20 2010, DIYbio google group |
| + | |
| + | |
| + | "Methylene blue is available in crystal form, if you're worried about |
| + | degradation over time. A little goes a LONG way." |
| + | :: -- Simon Quellen Field, Aug 20 2010, DIYbio google group |
| + | |
| | | |
| == Quick and dirty ways to do gel extraction of DNA == | | == Quick and dirty ways to do gel extraction of DNA == |