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

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