DNA extration - questions - (Aug/06/2006 )
I would like to know what is the function(s) of the following chemicals in gDNA extration?
in this experiment we are aiming to isolate gDNA from abalone (Haliotis midae)
- Tris is a buffer, used to maintain pH.
- CTAB is a lysis reagent, used to burst the cells.
- beta-mercaptoethanol is a a reducing agent and acts as a protein denaturant.
- EDTA is a chelating agent, used to bind free 2+ and 3+ ions. It is included to bind Mg++ particularly, because this ion is needed by many enzymes that might degrade your DNA if there were magnesium available.
- Chloroform is an organic solvent which is immiscible with aqueous solutions. It's likely you'll be using it to clean up your crude extract, as the DNA remains in the aqueous phase, while other things (proteins, lipids, charbohydrates) will be retained in the chloroform phase.
- Ethanol is an alcohol in which DNA is difficulty soluable. You'll likely be using it to precipitate your DNA, to further purify it, and to reduce the volume of liquid it's suspended in.
i am not sure but i think the carbohydrate wil not be dissolved in chlorophorm. so, the wash with 70 % Ethanol helps to get rid of them...is that right??
On further thought, I think a lot of this junk stays the the interface between the aqueous and organic phases.
Carbohydrates are not very soluable in ethanol; we routinely use ethanol precipitation to recover them. Perhaps they're soluable at %70, though...