Pretty much every standard DNA stain (GelRed, GelGreen, SYBR green, EtBr, etc...) can be used this way: directly added to the loaded sample.

For routine PCR we generally use GoTaq (Promega) where the stain is already fix included in the PCR mix.

However, for more sensitive DNA size evaluation the only unbiased way (to my knowledge) is to run the electrophoresis entirely stain-free and then do post-run staining of the gel.

Hi,

In my lab we use GelRed (http://www.biotium.com/product/product_types/search/price_and_info.asp?item=41002). We prepare a dilution 1/300 with water and mix 2-2.5,5 uL of this dilution with PCR product (3-5 microL) and 2-2.5 uL of loading buffer. Finally, we load this mix in the well. The only problem is that GelRed seems to modify the migration pattern of some fragments. In our case, this aspect is not so critical because we are interesting in presence or absence of the band.

Hi,

In my lab we normally use EtBr for gel staining and yes it may be dangerous but if used properly with good gloves and lab coats it is perfectly safe. But you can also try GelRed as mentioned by Jose Manuel Estivalis or maybe GelGreen

In our lab we use either EtBr or gelred. Both agents need to handled with care, allthough EtBr is more carcinogenic. We use a 1/50.000 dilution of EtBr or 1/30.000 of gelred in our agarose gels, after it has cooled down. We then load 10 ul of our PCR product or 5ul pcr product with 5 ul water, and 3 ul of Orange DNA loading dye.

Good luck!

In our lab, generally we use EtBr, but we have also used green view dye (from chromas), that gives good result, and it is easy to handle. It is safe and we didn't need to dilute it, we use it directly with samples, and it can be viewd without transilluminator.

Keep in mind that DNA in the case of mixing with the dye in front of the gel result in different gel pictures. So what I want to tell you is that DNA with different dye run different. So we allways let first run the DNA and then poststaining in front of making the picture.

Thanks - I have access to EtBr, and am hoping to avoid it. I prefer to co-load with my sample as there is less background noise than with the stain mixed in the agarose. Also, I am doing small (10µL) PCR reactions to minimize my amplification cost (using Phusion polymerase) so don't want to waste 5µL of the product in the visualization process. With the SYBR Green, I only had to load 1µL of product.

I have been using EtBr for PCR product staining for the past ten years. I am also trying to avoid it since it is dangerous. To make my DNA band in the gel shining like moon light, I normally stain and destain the gel for longer period of time.

Pretty much every standard DNA stain (GelRed, GelGreen, SYBR green, EtBr, etc...) can be used this way: directly added to the loaded sample.

For routine PCR we generally use GoTaq (Promega) where the stain is already fix included in the PCR mix.

However, for more sensitive DNA size evaluation the only unbiased way (to my knowledge) is to run the electrophoresis entirely stain-free and then do post-run staining of the gel.

EtBr cannot be used mixed to the loaded sample since it migrates to the opposite direction of the DNA migration.

Just try it ;-)

The loading dye will intercalate in between the double starnded DNA and it will move together with the DNA from the negative pole to the postive pole.

Dear, Linda

I suggest to try it , carefully

sorry mam i do not have experience with PCR

Yes you can use Midori Green Direct in 1:10 dilution. 1ul stain and 10 microliter sample or ladder. Apart from this you can dilute the your stain to 1:1 concentration and then later on you can use it in 1:5 dilution.

The best thing for you to do will be to run the DNA first and then stain prior to taking a picture. I have always done it this way and it worked well for me. I agree with Sven that DNA run is specific to the dye used. Good luck.

We use GelRed (4:1000 dilution within the loading dye). It works well but yes if you need exact size evaluation it has somme influence to the migrating DNA (even dependent on the DNA quantity).

There are a number of stains that can be added directly to any DNA samples including PCR products. They work very well.

The two I have tried are RedSafe by InTron and EZvision by Amersco.

RedSafe stains proteins too and a very high molecular weight band at the top of the gel corresponding to Taq will be seen on gels.

Note that Ethidium Bromide is toxic and mutagenic but the alternatives may be just as toxic and mutagenic even though they may have passed the AMES test.

These dyes are desirable because they say mostly in the gel and there is no powder handling.

However the dye stocks are fairly high concentration so it would be unwise to get it on your skin as any DNA binding molecule has mutagenic potential.

I have used EvaGreen (not sure who makes it but available from Jena Bioscience) like this - works pretty well. They even sell pit pre-mixed with ladders so your ladders fluoresce without any extra stain too. Its not as sensitive as post-staining with SYBR gold (both fluoresce in blue light / 488 nm laser) but most things are not as good as SYBR gold. I haven't directly compared to ethidium in the gel.

I think you should refer to this link:

https://www.researchgate.net/post/Can_anyone_suggest_alternatives_for_ethidium_bromide_in_terms_of_a_non-toxic_DNA_staining_dye

It all depends on how tightly the dye binds to DNA. For SYBR green the binding is to all intents and purposes covalent and fragments behave as if they were stably labelled. For Ethidium bromide the binding is less strong, so the dye equilibrates with the buffer solution. When EtBr-bound DNA comes into contact with ETBr-free buffer, the DNA begins to lose EtBr and its electrophoretic mobility changes. The result is unpredictable and quite often messy. The solution is to add dye to the gel and buffer instead of the sample. It produces a lot of dye-contaminated waste and gives background fluorescence, but it works.