Gel without ethidium - (Aug/29/2007 )
I am planning to purify the digested plasmid (9.6kb) for the first time, so I am wondering whether it is essential to gel purify in the gel without ethidium, if so how much concentration is used to stain the gel briefly. Also what concentration of agarose gel is used? Please reply at earliest
you need ethidium in your gel to see your DNA under UV when you will cut the DNA.
use 5 ul of 10 mg/ml ethidium bromide for 200 ml TAE buffer
concentration of agarose gel is depent on your experiment/purpose
I have used the gel both with Ethidiumbomide and without Ethidiumbomide (staining after gel-electrophoresis) for colning propose. They are not different for visualization of DNA under UV. Instead, Ethiduumbomide helps to protect more or less DNA structure during the time it is examed under UV. The concentration T. reesei have told is recommened and you should stain the gel between 15-20 min.
i think T reesei concentration is 2.5x too much concentrated.
I will be using it for cloning purposes by ligating the purified plasmid with the purified insert. I had heard that ethidium bromide influences the superhelicity and also it s electrophoretic mobility. I am not sure whether to use it before or after the gel run. Also if used after what concentration to be used?
Well, the main task of Ethidium bromide in this matter is to visualize the DNA under UV light. And of course, it will have to alter the native structure of DNA by binding to it. Therefore, Ethidium bromide may be a very strong mutagen, and may possibly be a carcinogen or teratogen and so on.
I would suggest that you should perform sequencing experiment after you succeeded with cloning as I usually do.
In case you want to stain your gel after gel-electrophoresis, you can do it in variant concentration by dilution the Ethidium bromide stock (10mg/ml) from 1:10,000 up to higher concentration, which is depent on your DNA concentration, time and how you handle and manage the Ethidium bromide waste. The higher concentration may help to protect the sticky ends, which are sensitive to UV light as well as they may be the most important key of your experiment. Additional suggestion, you may as well protect you DNA from be damaged when it exposed to UV light by addition of cytidine or guanosine 1mmol/L to the electrophoresis buffer when you prepare your gel.
You don't *have* to use ethidium bromide - there are other dyes. For vector preps, where you are digesting 1ug or more DNA and elimination of the UV step is desriable, I would recommend methylene blue. See point 5 in this article
Thanks for prompt and useful response !!
I would suggest that you should perform sequencing experiment after you succeeded with cloning as I usually do.
In case you want to stain your gel after gel-electrophoresis, you can do it in variant concentration by dilution the Ethidium bromide stock (10mg/ml) from 1:10,000 up to higher concentration, which is depent on your DNA concentration, time and how you handle and manage the Ethidium bromide waste. The higher concentration may help to protect the sticky ends, which are sensitive to UV light as well as they may be the most important key of your experiment. Additional suggestion, you may as well protect you DNA from be damaged when it exposed to UV light by addition of cytidine or guanosine 1mmol/L to the electrophoresis buffer when you prepare your gel.
A high concentration of ethidium bromide will not protect the DNA in any way, and in fact will lower the cloning efficiency by interfering with the ligation reaction.