I almost always have used EtBr to visualize fragments for cloning- in my opinion the greatest disadvantage to using EtBr is the risk of damage to the DNA due to using UV for the visualization. For this reason, in one lab that I worked in, Methylene Blue was used to stain the DNA before gel extraction, as then the DNA was visible using visible light.

Hello,

I always use EtBr staining when I have some cloning to do, and it always works fine.

But I have to admit I never wondered if EtBr remains after extraction. I guess the answer is no.

yes it is reliable to stain the digestion product by EtBr for gel extraction before ligation. However, the UV used to examine stained products can preturb ligation success (by causing thymdine radicals and dimerization). Therefore, minimize UV exposure by excising your band quickly - make you cuts with UV on - turn it off - excise the cut block containing product for subcloining

I almost always have used EtBr to visualize fragments for cloning- in my opinion the greatest disadvantage to using EtBr is the risk of damage to the DNA due to using UV for the visualization. For this reason, in one lab that I worked in, Methylene Blue was used to stain the DNA before gel extraction, as then the DNA was visible using visible light.

I always extract EtBr-stained bands for cloning. I think that during the purification as you have high concentration of denaturating agents as guanidine hydrochloride the EtBr goes away. But it should easy to test if EtBr is still there. You could apply an aliquot of your extraction in agarose gel and, without stain it, try to visualize the band at 302 nm irradiation.

Qiagen claims removal of EtBr, though I have not analyzed the DNA after extraction. EtBr does not interrupt ligation of DNA exposed to EtBr and subsequently "cleaned up", as evidenced by the decades long history of successful ligations using EtBr. Most/all "clean up" processes historically include an organic solvent step somewhere, and these type solvents are known to extract EtBr from nucleic acids.

Though this is speculation, I doubt ligase itself would be inhibited by EtBr to perform blunt- or sticky-end ligation, though certainly other molecular biology processes would be.

Ligate away!! :)

If you are looking for options try Syber Green. What can intervene is UV, try to cut the bands as quickly as u can to avoid any dimerization.

I agree with Scott and Donna and others- I always use EtBr to visualize fragments that I cut out of a gel and purify, whether for ligation or sequencing or other application. The major problem can be UV-induced damage, and minimizing exposure, especially to short-wave UV, is best. If you have a transilluminator with rheostat or just a low intensity setting, that works well. But still, I try to keep the exposure to UV to a minimum by working as quickly as possible. I believe that John is correct with regard to extraction procedures containing solvents that should remove at least some of the EtBr, but do not know how much.

Hi Amir

There is a gel elution column (minus EtBr) that claims removal of EtBr during the process...you can go for the same...or else as suggested above by many researchers minimize the UV exposure during DNA isolation from gel.....

bye

I made a transilluminator using leds that emitted at blue range. Then I use cybergold stain. This eliminates exposure to UV.