EtBr in Gel Electrophoresis: To Add or Not to Add?

[indoubt]

should i add EtBr in the gel solution, when i run the mRNA?

thanks!

 

[Monique]

yes, a 1.5% agarose gel should work with EtBr

 

[iansmith]

When adding EtBr in your gel it should be could and you should add some EtBr in the positive well. EtBr is positevely charged and it will move out of your gel during the electrophoresis leaving a dark spot at the bottom half of your gel. This is not good if you want to use your results in publication, poster or seminar.

Adding EtBr only in positive well works fine also. You use less EtBr and minize the amount of contaminated material.

 

[Moonbear]

iansmith, when you add EtBr only to the well, what ratio do you use with your sample? I have always just added it to the gel (that's just how I was taught and never gave any thought to whether it needed to be done that way). If it works just to add it to the well, that sounds like a good solution, especially when I pour a gel and cut it in half (only need half for a few samples, but then save the other half in case something goes wrong and I need to run it again). With the leftover half, I often end up having to just throw it away because I don't need it, so no need to have it contaminated with EtBr if I don't have to add it to the gel itself. I'm always interested in learning new ways to reduce contaminated material. Our lab is doing much more work now generating our own probes for in situ, so this is pretty timely to address.

 

[iansmith]

We usually add 5 uL to well and it is good enough for one run. I have see people in our lab add up 20 uL and used for 3-4 runs. I don't like adding a lot because EtBr is inactivated by light and has the stock get older, it is not as stable as a fresh stock.

The only material that you do not get contaminated will be the flask you used for pouring the agarose gel.

 

[indoubt]

hi guys!

thanks for you reply, but i am asking about mRNA. EtBr will stick into the double stranded DNA, but since mRNA are single stranded then how come EtBr stick into it so we can visualize it with UV light? so should i add EtBr when i run the mRNA?

thanks again!

 

[Moonbear]

I was just discussing this with a post-doc today. EtBr won't stick very well to RNA, but we're hoping to get enough signal just to confirm probe quality. One approach we discussed is running it on a thin acrylamide gel instead of an agarose gel and then soaking the gel after it has run in solution containing EtBr. We're not sure this is going to work (no one in my lab is a molecular biologist, so we are learning these methods as we need them). I'm not planning on doing this right away though, so if you find a solution to the problem first, let me know!

 

[Monique]

It works for this company, it should work for you http://www.biosupplynet.com/cfdocs/...on=/8398/embigels.gad&supp_id=8398&deal_id=48

 

[Moonbear]

Thanks Monique! It's amazing to me they are trying to sell pre-cast gels. They are so simple to make, I just can't see paying more for someone else to make them. There are a lot of things in molecular biology that are nice to get in kits, especially if you don't use it often enough to want to have lots of stock reagents around, but even my lab knows how to make gels (though, apparently I'm the only one who had trouble with the definition of "cool enough to touch the flask" for knowing when to add the EtBr and pouring the gel...I'm the only one who can swirl the flask to mix the agarose into solution without insulated gloves...but even I've learned the feel of the right temperature and can pour a good gel...then again, I'm not yet old enough to be one of those profs the students chase out of the lab...I've worked for those, the ones who make a big mess when they come into the lab, so everyone chases them out as quickly as possible if they get the urge to do some benchwork).

 

[Monique]

then again, I'm not yet old enough to be one of those profs the students chase out of the lab...I've worked for those, the ones who make a big mess when they come into the lab, so everyone chases them out as quickly as possible if they get the urge to do some benchwork.

lol, that's funny

I guess the pre-cast gels work for people who run many agarose gels on a daily basis. I always wondered how much of the EtBr is evaporating when added to the hot agarose (especially when remelting gels). But yes, why waste money.. it's not much of an effort to melt 1g of agarose in 100 ml of buffer

 

[iansmith]

it's not much of an effort to melt 1g of agarose in 100 ml of buffer

It much more effort to prepare a large batch (500 mL) and keep it in 65-70 C water bath.
I guess the effort is that you need that had EtBr later.