6 replies to this topic

[mad]

Hi everyone,

i'm new to the forum so please bear with me

I am starting on a new electrophoresis project and have trouble deciding the method of staining my gel. Is it possible to stain the DNA sample with the etbr before i load it into the gel?

Thank you for your help

MAD

[chrisbelle]

mad, on Apr 6 2009, 02:39 PM, said:

Hi everyone,

i'm new to the forum so please bear with me

I am starting on a new electrophoresis project and have trouble deciding the method of staining my gel. Is it possible to stain the DNA sample with the etbr before i load it into the gel?

Thank you for your help

MAD

Hi there,
It's better to directly stain the gel with EtBr (add to gel after cooled, before pouring into cast) or do stain in EtBr solution after you run gel, but you need to destain it. There are new methods to stain gels, for instance GelRed, SYBR Green and stuff like that. There's a new one where it is already mixed into the PCR buffer, and you can directly load onto unstained gel. Choose the method best for you, but we like to directly stain with EtBr in our lab.

Cheers,
Chris

[noelmathur]

I second Chris. If your boss has got money, avoid EtBr altogether. if you have to use EtBr, First make a stock of EtBr and then use it in the liquified agarose, once temperature is below 45°C. (if you can hold the flask in your palm for 20 sec.s thats the time to add EtBr, otherwise it evaporates and stains the band weakly)
Avoid using staining later in EtBr buffer (i.e buffer mixed with EtBr) First of all its very messy and second is, you tend not to get sharp bands as you may want such bands sometimes.

[hobglobin]

noelmathur, on Apr 6 2009, 11:23 AM, said:

I second Chris. If your boss has got money, avoid EtBr altogether. if you have to use EtBr, First make a stock of EtBr and then use it in the liquified agarose, once temperature is below 45°C. (if you can hold the flask in your palm for 20 sec.s thats the time to add EtBr, otherwise it evaporates and stains the band weakly)
Avoid using staining later in EtBr buffer (i.e buffer mixed with EtBr) First of all its very messy and second is, you tend not to get sharp bands as you may want such bands sometimes.

But EtBr does not evaporate. Perhaps the vapour is carrying some EtBR (i.e. aerosol) out of the flask (therefore I do it under the fume hood). But not enough to weaken the staining.
And some people prefer the staining after the run this thread is interesting on this, views or philosophies differ in this topic....

[mastermi]

Views differ in this topic. That's right.
I do prefer staining the gel after the run in EtBr-solution. We have one room for our whole institute with an EtBt-bath and the UV-chamber. This is the only room where we have EtBr contamination. Everything we work with in our personal labs is therefore EtBr-free (like the gel-slide and the chamber)...

[PhDs]

Biologists:

I would like to  disclose a very important fact: "Goldview" DNA stain from Beijing (China) SBS (http://www.sbsbio.com/) is actually Acridine Orange.


So if you are using "Goldview" instead of  Ethidium Bromide, you should know "Goldview" is  Acridine Orange, which is available at very low price from Sigma or Invitrogen (as low as $23.2/5g).

Acridine Orange is also mutagen/carcinogen. Many scientists in China and around the world may have exposed (without wearing gloves) to this so-called "safe" compound purchased from SBS.

The structure of "Goldview" has just been identified by a company in California. Data is available upon request (after signing NDA).

More about Goldview's advertisement:

http://www.sbsbio.com, http://baike.baidu.c...iew/1650421.htm, http://www.protocol-...osts/39211.html,

http://www.biocompar...tive-to-EB.html,

http://www.biomaxkor...olbio/stain.htm,

http://www.wrc.org.z.....n 07/2046.pdf, http://www.google.co...Cj8G4cma_bTt4uw

http://www.geneshun...... Products.htm, http://www.reference...5/CCLM.2005.141,

http://www.springerl...6275m006665w58/

http://lib.bioinfo.pl/meid:175960

Edited by PhDs, 08 May 2009 - 06:04 PM.

[swanny]

Oh boy, here we go again. The first posting is fair enough, but then we rapidly spiral into the old "how dangerous EtBr is so use a 'safe' alternative (which often turns out to be more toxic than EtBr)".

EtBr has been used as a treatment for bovine parasites, being injected into cattle at 1 mg/kg. Thinking about your average cow, that's a heck of a lot of EtBr. Have there been reports of mutant cows? No. Have there been reports of mutant farmers who eat said cows, or drink their milk, or their blood (the farmers are Masai:- apparently it's quite a nice drink go figure). Again, no. Have there been reports of either cows or farmers getting teratomas? No. Even taking the racially adjusted nature of many Western news 'services', EtBr isn't the bogie-man it has been made out to be. Having said, you'd be a fool to work with it without wearing gloves, right? Be professional, be careful.

Rant over.

Hey mad, you can choose to add the EtBr into the gel, or in a post-gel staining bath. Don't add it to the loading buffer, because it could cause the fragments to migrate slower, especially as the EtBr goes in the opposite direction as the DNA.