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Non-hazardous substitute for ethidium bromide? - (Aug/15/2012 )

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Is anyone using Gel red for gel electrophoresis?

I needed to ask something about this product so if anyone has any experience in using this then please let me know.

Moved and merged so as to keep recent discussion in one place. Bob

-csadangi-

Yes I use it, csadangi, what is your question?

-leelee-

i add 5 uL of gel red to 50 mL of TBE buffer when its hot.
i use the VWR 6X loading dye.

But when i run the gel i lose the loading dye. Is it caused due to the gel red?
I did it with SYBR and it worked fine there.

-csadangi-

the dye in the loading buffer is a tracking dye. you use it to judge when to end the run. if you are losing the band during the run then it is probably diffusing and not due to the visualization dye. you may be able to see it if you look at the gel on a white background (we put white paper under).

-mdfenko-

RE: csadangi:
As to why loading dye is lost when using GelRed in TBE?
DO you mean that the dyes in the loading buffer disappear during electrophoresis?
What dyes are in your DNA loading buffer? xylene cyanole and bromophenol blue?
Have you checked the pH of your running buffer before and after use?
It can change after use. And of course if it is not slightly alkaline (pH 8) to begin with,
the dyes can change to lighter colors.

See my earlier posts above. We now no longer use ethidium bromide.

Also, we pour our gels without any dye in them and without any dye in running buffer (see recipe in my earlier post above).

We also no longer use GelRed as it sometimes creates migration artifacts for some of our PCR assays and sometimes causes
smearing. GelRed is also classified as hazardous waste, so is less convenient overall. GelRed is VERY sensitive, giving very
bright fluorescence (UV 302 nm transilluminator). So, it looked promising at first, but now we use EZ-Vision instead.

We extensively tested using GelRed diluted 1 in 2,000 in our 6x DNA loading buffer, adding 5 uL to our 25 uL PCR.
Then loading 10 uL into gel. But the used gels now end up with GelRed in them so have to be discarded the
same as EtBr haz waste. Which is something we want to avoid (we are a diagnostic lab, short staff and pressed for time).

Also, GelRed causes some amplicons to migrate at different rate than expected. Regardless if used in gel or in DNA loading buffer.
Again, not good for a diagnostic lab. So we stopped using GelRed.

Finally we discovered that EZ-Vision (Amresco) performs same as EtBr. That is, no migration artifacts, no affect on migration rate
for any of our PCR assays and same band intensity and detection limit as EtBr. One difference to note is that EZ-Vision needs to use
UV transilluminator set to 365 nm (whereas EtBr uses UV at 302 nm).

We use EZ-Vision concentrate (supplied as 5 mL dropper bottle) diluted 1 in 100 in our 6x DNA loading buffer.
We add 5 uL of DNA loading buffer to our 25 uL PCR, mix, load 10 uL per well. Bands migrate precisely as expected (we ran many
different PCR assays, those whose migration were affected by GelRed migrated as expected when using EZ-Vision in DNA loading buffer.

We consider EZ-Vision as non-hazardous (because Amresco supplies plenty of documentation and studies
backing up their claim). So we can discard used running buffer down sink with running tap water and we can dispose used gels
(double bagged for smell, due to TAE which airborne yeast will ferment) in our regular waste.

For our understaffed diagnostic lab that counts every penny we spend, this is a big deal. Our staff time is valuable.
The hazardous waste disposal costs us $100 per 25 L pail of used gels and EtBr "tea bags".

I will post a few photos and more detailed recipe.

-Falco79AD-

Here are 2 gel photos. One gel has EtBr in it and the photo was taken on UV light box set at 302 nm. The other gel does not have any dye in it. Instead the DNA was loaded using 6x loading buffer containing 1/100 EZ-Vision stain. This gel photo was taken on UV light box set to 365 nm.

The 6x loading buffer consists of 1x TAE (pH 8.3), 50% sucrose w/v, 0.1% xylene cyanole and 0.1% bromophenol blue.
Add 10 uL of EZ-Vision concentrate (Amresco cat# N391-5MLDRP) to 1 mL of the 6x loading buffer. Keep in dark (use amber tubes). Remember to also use this for your size markers.
Attached Image

Attached Image

-Falco79AD-

you mentioned "We use EZ-Vision concentrate (supplied as 5 mL dropper bottle)"
I did not find the product like you described above in the amresco website, could you tell me the cat number?
thanks,

-hvripara-

hvripara on Wed Dec 26 02:57:08 2012 said:


you mentioned "We use EZ-Vision concentrate (supplied as 5 mL dropper bottle)"
I did not find the product like you described above in the amresco website, could you tell me the cat number?
thanks,

just look at the post immediately before yours

-mdfenko-
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