I'm running my pcr products in an 1% agarose gel made with TAE buffer. The volume i use is 20 ml with 1ul EtBr. I attach a picture. I want to get as dark as posible for the background and as bright as possible fo my bands.
Please HELP!! :(
You can use Gel Documentation instrument. It gives you nice photo for your bands.
In addition, you can also add EtBr. to the buffer in the tray to increase the visualization of your bands.
Try to increase the concentration of DNA (eg use more DNA/narrower wells); the image may also indicate that the agarose was not thoroughly mixed with EtBr.
you can use light washing in buffer for 5-10 minutes without etbr to wash away the etbr in this gel but generally just use less etbr in your gels and make sure that the buffer in the gel tank is fresh and the background will be much lighter
I'll try putting the etbr in the loading buffer... and if that doesn't work i'll try washing the gel. I always use everything fresh so one less thing to try haha. Thanks!!!
Hola Agustina, lo que podés hacer después de la corrida, es sacar la primer foto y poner el gel a lavar en una solución de MgSO4 1mM, por 5 min-10min, en lo posible con agitación. El proceso se puede repetir varias veces hasta limpiar el fondo. Te recomiendo sacar una foto por cada lavado. Saludos, Mariana
It is due to the inefficiency of your gel documentation system may be......
Low quality agarose....
ETBR you have to add in agarose solution before casting
Your gel looks fine.But your had captured the image using a normal camera.Use a sophisticated gel documentation system.It may have the software which can convert this picture dark as u wish.
OR you can download one like this
https://www.thermofisher.com/in/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/e-gel-electrophoresis-system/e-editor-2-0-software.html and use in your PC for further editing your gel.
Regards
Noble
i wish i could use some documentation system. All we have is a dark chamber, a transilumminator and a regular camera :(
The problem is not your documentation system. I used one similar to yours more than 20 years ago (with a polaroid camera).The problem is EtBr itself. EtBr doesn´t give you bright bands without adding background noise, too. My suggestions are: 1) You could try and wash your gel after electrophoresis in running buffer for 15minutes. This would reduce your background but probably the intensity of your bands, too. 2) You could also stain and destain your gel after electrophoresis for 15minutes each. 3) Get rid of EtBr, even though it´s extremely cheap, and use another gel stain that doesn´t give you that much background.
best regards
Dirk
To me it looks as if the ethos I bromide is not being mixed properly. When are you adding it in? In our lab we add the EtBr after letting the gel cool down for 1 'minute and swirling to thoroughly mix.
how much DNA are you adding and what sized wells are you using? Try upping the volume you are adding next time.
Also, unless you absolutely need the gel to be at 1%, I've had much better experiences with 2% with Ethidium bromide staining.
EtBr runs in the opposite direction of your DNA (i.e. towards your sample slots). That´s the reason why high molecular weight bands sometimes have a high background wheras low molecular bands in the same lane appear brighter and have a better contrast.
It doesn´t matter when you add your EtBr. In the past they told me to let the agarose cool down to 50°C before addibng EtBr. But then, for many years, I added it right after taking the agarose out of the microwave oven. No difference. You could even recycle an old gel with EtBr, melt it again in the microwave oven and pour it again. There would still be enough EtBr left intact to make your DNA visible.
I would still suggest another DNA stain for your gel. They tend to be more expensive but the result is worth it. At least they are cheaper than buying a high-end documentation system for your gels.
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