Dear Raymond Fernalld,

If it is possible, you may compare three different dyes in agarose gels with the same source of DNA thus you could easily see whether the problem comes from the dyes or not.

Best wishes

Dear Raymond,

We also have similar problems in our lab when using GelRed. Specially sensitivy/visualization problems with well know extraction protocols. We substituted ethidium bromide for GelRed about a year ago, and started using it in a higher dilution than recomended (which works fine for PCR reactions). To improved these issues, we started using GelRed in the appropiate dilution and we added RNAse A to samples. All of this was particularly useful when working with complex reactions mixs (like "in house" miniprep protocols, without purification steps using columns).

On the other hand, we tested Sybr safe and reach better results.

I haven't tried GelRed for gel extractions but its a bulky dye. How big are the fragments you are trying to electrophorese? I use GelRed cast in gels at 0.5x for products >500 bp, but any PCR products smaller than that it affects the mobility and you get smears. Still on SYBR safe for small PCR products. GelRed is also a lot more sensitive than ehtidium/SYBR, which can make PCRs look smeary when its just PCR bands you can't see with EtBr.

Thank you to everyone who has responded thus far. The fragments we are generally trying to electrophorese and isolate are in many cases larger ones, in the 8-10 kb range. However, we have had issues with extraction of fragments of all sizes.

Hi Raymond

When we started using Gel Red we also observed the smearing problem. We solved it by consequently diluting the sample DNA. As for example if I loaded before 5ul of a PCR I now only used 1ul. However before loading I diluted it back to a total volume of 5ul or 10ul (can be easy done by changing your loading buffer dilution). Using this step we hardly observed any smearing anymore.

We also had low yields. I personally blamed the high sensitivity of Gel Red for that. Which means what looks like a fat band (lots of DNA) on a Gel Red stained gel is a faint band on a EtBr gel. As you expect a lot of DNA from the fat band you might end up disappointed. To prove or disprove this hypothesis you could follow Ebru Tekin's suggestion. I personally decided to not care so much about it as I never had good yields with other dyes either and gave up on my search for good gel extraction method/Kit. I rather try t avoid this step as much as I can.

Hope this Helps

Berni

 Berni is right, I observed the same problems when switching to GelRed. 5 ul of amplicons was far too much and caused very aberrant bands. The product literature states 10-20 ng sample DNA per band and it is very important not to exceed this, but can be difficult if you don't have accurate DNA quantitation at your disposal. I now use 1 ul amplicons, 1 ul Gel Red and 4 ul 1x TAE and it works fine.

HI everyone,

i'm using gel red in post staining, and sometimes i find a different staining of teh gel from left to right...i think it could be related to the shaking during staining, but not sure. We use an orbital shaker whic always move in the same direction, so it could be involved in that effect. We use it at 80 rpm, and i found that if you shake slowly (ie 50 rpm) the staining is not good. We use a 3x gel red stain as propsed by the manufacturer instructions. But many other times it works very good, so may be it has percipitated, so i will heat at 45ºC and shake in order to redisolve, just in case. But i would like to know if someone has found problems in post staining using gel red.

Thanks and kind regards

We had simmilar problems. We realize that RedGel was loosing volume by keep it at room temperature, you know RedGel is already in water and the heat was drying it.

We solve the problem keeping it a 4ºC and just taking it out when we use it.

I hope it will help you.