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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 ==