dna purification - (Feb/13/2007 )
now i am trying to purify herpesvirus dna from cell culture. i used sinzger method. but unfortunately, i have no band at all after cutting by restriction enzyme. i do not know where is the problem.
i wait untill CPE is 100% then collect the media and the cells, then centifugation and washing twice.
then i used C1, nuclease enzyme, digestion overnight and phenol purifecation and ethanol precipitation.
so, please if you have an idea or recommendation please tell me.
the digestion over night is with the rnase???. In general for DNA purification, you will need a lysis buffer (with sucrose, a detergent), add protease (they work at more than 55C so check this carefully and with 1hour of incubation, if working with a lot of cells), then let it cool to 37C and add Rnase incubate for an 1hour. at this point you could do 1 of 2 things:
1. Phenol/chloroform extraction and the alcohol precipitation steps that are explain below.
2.Add a high concentration salt buffer(1vol) (could use NaCl in water until it almost out of dissolution (I think that is around 4M) and filtrate it), incubate on ice(this step is very important!!!!!)for 5min and centrifuge at low-med speed to get rid of proteins and debris. Keep the supernatant, add to it 2-2.5 volumes of cold alcohol (isopropanol or absolut ethanol)(better if they were keep at -20C) and 0.5 volume of a salt buffer (eg sodium acetate ,Lithium chloride) mix by invertion (in this step usually you can see the DNA) and incubate overnight at -20C, then centrifuge at high speed for at least 15 min, decant the supernatant and wash the pellet with 70% ethanol (tap the tube and be sure to disolve the pellet) and centrifuge for 10-15 min, repeat wash steps, after that , get rid of all ethanol by air dry or use a savant if available. Finally disolve the DNA in 0.1-1X EDTA. Hope this help. oohhh before I forget, remember that you are searching for virus DNA and it is smaller than cells DNA so watch the rpm and time in the centrifuge.