phenol/chloroform extraction.... - protocols for large/medium scale (Sep/11/2006 )
hi there, which protocols should I use for extracting high ammounts of DNA from Trout sperm??? 
the only thing that comes to mind is to use a guanidine thiocyanate + detergent lysis buffer. Followed by a RNAse step, then 3 or 4 phenol/chloroform extraction.
As all the component are self prepared, you can easier scale up the reaction without end. This is also superior to DNA binding kits as it is far cheaper, however it takes more time. (about 4-5hr) and as a result there are more chances of external nuclease contamination. So you have to be careful and perhaps work in a cold room (to be extra sure).
Does this fit your requirements?
which temperature is suittable for the cold room, to expect no action of nucleases?
My objective is to prepare a rainbow trout DNA solution, and by this way replace (locally) the commercial Salmon Sperm DNA saving some money.
I will be grateful if you can give me any other recommendation/s.
Thanks!
Diego
Wait phenol/chloroform procedure is only to purify DNA or inactivate enzymes, and this treatment is the last step extracting DNA from maxiprep.
which temperature is suittable for the cold room, to expect no action of nucleases?
My objective is to prepare a rainbow trout DNA solution, and by this way replace (locally) the commercial Salmon Sperm DNA saving some money.
I will be grateful if you can give me any other recommendation/s.
Thanks!
Diego
No, the (guanidine thiocyanate, detergent lysis) buffer does not generate a 2 phase solution. It is a monophasic solution. Add it to a cells without cell walls, and the solution breaks down the cell walls, denatures the protein, degrade (to some extend) the RNA and solubilise the lipids. A magic solution, which however isn't perfect. There is residual protein and RNA.
Do you need the recepie of this solution and a basic protocol?
Working in the cold, helps as it slows down whatever nuclease is around, giving you more time to clean up the DNA. So any low temperature would do really. The real test is after the DNA is prepared and place it in long term storage. If the prep was no done well, the DNA will be gone after a few months. Done correctly and it'll sit at room temperature for a year with little degredation (though I won't recommend you do this!)
I would recommend a cold centrifuge too.
Although I must ask, is salmon sperm DNA that expensive? Or might you simply have access to lots of Salmon sperm for some reason or another. And may I ask, what do you do with Salmon Sperm... I know it is used as a carrier... but I haven't seen it used that often.
that is correct. Phenol is used to denature proteins (although archean proteins can be resistent to phenol denaturation), which centrifugations pellets down onto the interphase.
Thanks for your tips! I hope they will be helpful. And, as regards to your question, in my lab we use to make DNA extractions dayly. Lots of people doing DNA extractions, and quantifying ir. There is also part of the group using it in Southern Blots, and some other people uses S. S. DNA in some stuff related to TOLL proteins, but I do not know what do they do, exactly.
thanks a lot again. I´ll tell you my results!!