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What causes surface fluorescence on agarose gels?

agarose surface fluorescence smudge

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6 replies to this topic

#1 Falco79AD

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Posted 13 August 2012 - 07:15 AM

We add ethidium bromide or GelRed to molten agarose in 1x TAE prior to pouring our gels. Sometimes the gels have odd random, smudge type pattern scattered all over the surface (see photo).

Our gels can be used same day they are poured or can take 2 weeks to use it all up. When not in use we store the gels in walk-in fridge to prevent yeast growing in the 1x TAE buffer. We use 7 rows of 20 well combs in 15 x 25 cm gels.

It appears the smudgy surface fluorescence eventually disappears after several days and several runs (we use the bottom rows of wells first, and work our way up one row at a time).

We compared adding the EtBr or GelRed to very hot molten gel and cooler molten gel. So far we have not seen any pattern suggesting what may cause the appearance smudgy surface fluorescence.

Has anyone else experienced smudgy surface fluorescence on their agarose gels?
If so, any ideas why it happens? ... Remedies?

Thank you,
Andre

Attached Thumbnails

  • 12-Aug 10 GelRed 1in4 in gel 500 msec.jpg


#2 hobglobin

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Posted 13 August 2012 - 07:46 AM

perhaps the EtBr wasn't dispersed evenly in the agarose, i.e. more agitating necessary when the EtBr is added?
First it appeared like a film of very tiny condensed drops to me (just like you have when getting stuff out of the fridge when air humidity condenses on it), but after electrophoresis it's not likely
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#3 Falco79AD

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Posted 13 August 2012 - 10:27 AM

We are very mindful to vigorously swirl the molten gel for several seconds after we add the EtBr.
We see the EtBr disperse throughout the molten agarose.

BTW, we add 25 uL of 0.2 ug/uL EtBr to 350 mL gels.
We also tried adding 200 uL of 1/8 diluted EtBr. ... stil happens ... apparently randomly.

We store the EtBr in the dark. When not in use we store the gels in the electrophoresis chamber in the dark on a cart in walk-in fridge
(we cover with a heavy green plastic grocery shopping basket). Likewise, the 1x TAE contains EtBr and we store i in walk in fridge in dark.

We even arranged so that only 1 person (me) pours gels for 2 months.

What's frustrating is that the surface fluorescence smudge seems to appear randomly.
Some gels are perfect. Some have surface smudge.

This ought to be a simple thing to figure out ... alas, it's still a mystery.

#4 leelee

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Posted 13 August 2012 - 06:23 PM

Do you carefully wipe down the transilluminator between taking photos, making sure to use a wet paper towel or something to wipe away any residue?

I ask because this very same smudging used to happen to a student in our lab, turned out she wasn't wiping the transilluminator after removing her gel so any buffer and moisture from the gel just dried onto the glass. This then showed up on her next photo.

#5 Falco79AD

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Posted 14 August 2012 - 04:56 AM

After every use we always wipe down the UV transilluminator with a damp spong using diH2O.

After talking more about this in our lab we think the smudgy surface fluorescence coincides with whenever
we get a swirly pattern on the surface of the gel after it sets. It may be that such gels were poured too hot.

We will try being more mindful of the temperature and pour the gel when it cools to about 60*C.
It appears that such gels have much less swirly pattern on the surface after it sets.
And we think this is when our gels also have no smudgy surface fluorescence.

I will try to replicate both, smudgy (poured when hot) and non-smudgy (poured when 60*C).
I'll post my results.

Thank you everyone for sharing your thoughts on this.

#6 phage434

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Posted 14 August 2012 - 02:56 PM

You may not be heating your agarose sufficiently, or for long enough. Agarose takes a while to dissolve, and will clump badly given a chance. Make sure the solution is crystal clear and well mixed.

#7 Falco79AD

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Posted 15 August 2012 - 11:16 AM

Thanks to all who have posted replies.
Our gels are thoroughly melted (no "ghosts of undissolved agarose) ... crystal clear.

Just now I posted another thread regarding non-hazardous substitute for ethidium bromide.





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