Glowing BLUE DNA Gels

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Mon Oct 3 12:02:54 EST 1994


Earlier this month I posted an article on the topic of blue gels:

| Regarding the on-again off-again thread about glowing blue agarose gels,
| I've been following the subject of "Glowing Blue DNA Gels" for sometime...
| ...Some people think that the source of the glowing BLUE is fluorescent
| additives or whiteners within laundry detergents that may be transferred onto
| your hands if you wipe them on your clothes or perhaps someone else's clothes.

Since then, I've had several interesting ideas and e-mail conversations 
with John Nash [nash at nrcbsa.bio.nrc.ca] and Scott Mulrooney
[Scott.Mulrooney at um.cc.umich.edu] about their observations of the
blue gel phenomena which I would like to pass along. Feel free to
comment on these and post any interesting observations of your own.

J> I have my own theory on blue gels also.  I find that I get them if I
J> use some TAE buffers and either run the gels very hot (high
J> volts/amps) for a short while, or if I run them overnight (low
J> volts/amps) without recirculating the buffer.
J>
J> I find that the blue creeps up the gel with time, from the bottom
J> towards the wells, and degrades the DNA in its path.  No amount of
J> staining/destaining gets the DNA back.  Recirculating the buffer seems
J> to solve this problem for overnight runs (and using a stirbar helps
J> for short runs), and so I theorize that the buffer is depleting in the
J> +ve chamber, and what's left degrades DNA (I should check the pH!!!).
J> 
J> I never see it with TBE buffer.  Also, it doesn't matter whether I add
J> EB to the gel or stain/destain as to whether I get the effect or not
J> (and I use very little EB in my gels).

S> ...I have run across gels with a bright blue glow when
S> in UV light. This has happened in gels that were run in TAE buffer
S> and run or stained in "old" buffer, that is 1X buffer that has been
S> lying around for 2-4 weeks or more. By using freshly diluted TAE,
S> I never had the problem. I also noticed that bottles of old 1X TAE
S> ofter had something that looked like mold adhering to the glass
S> on the inside of the bottle. It may be possible that these two
S> observations are related.... or maybe not.

P> There seem to be two observations with glowing blue gels. One is that the
P> places where people touch the gel (either TBE or TAE) glow. The other is
P> that the entire gel of the TAE variety glow blue under UV.
P> 
P> In the second case, your observations may be due to the same phenomenon -
P> maybe a breakdown or other conversion (precipitation?) of the buffer, or
P> perhaps a bloom of some microorganism (fungus?) in TAE buffers which
P> fluoresces and eats DNA by secreting nucleases (?).

P> John, Is the buffer you used the same as that of Scott?
P> ...and do you see this problem when the buffer is freshly made?

J> I never have tried it with fresh buffer.  My recipe is:
J> 
J> 1X is 40 mM Tris, 20 mM acetate, 2 mM EDTA, pH 7.8.
J> 
J> 					50X
J> 
J> 	Trizma HCL			228.8 g
J> 	Trizma base			66.4 g
J> 	Na acetate (anhydrous)		82.0 g
J> 	0.5 M EDTA, pH 8.0		200 ml
J> 	dH2O				to 1 litre.
J>                                        
J> I make it as 50X, filter-sterilize it into a sterile bottle, and
J> remove aliquots aseptically, but either dilute it fresh or dilute a 4
J> litre bottle and use that until it's gone - from days to weeks.  I
J> have not thought about whether freshly diluted or not-recently-diluted
J> buffer makes a difference.

P> Scott, Do you think this is the same thing?

S> Yes, it could be. I only see this with standard tris acetate buffer.
S> It does seem reasonable that buffer breakdown of some type may be linked 
S> to the blue gel observations. I have also corroborate John's observation 
S> of gels being run too long without buffer circulation leading to blue 
S> glow at one end. I don't have much experience in observing DNA 
S> degradation in gels run too long without circulation: this may result 
S> from nucleases, or from drastic pH changes at the ends of the gel leading 
S> to DNA degradation.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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