I have attached a CTAB protocol for which I am using for DNA extraction. I am not getting any supernatant on step 13 (page 2). After the addition of ammonium acetate, keeping on ice, and centrifuging, it is all just a clear liquid. Any idea what is going on? I am wondering if adding the isopropanol separately has something to do with it.
Dear Milo
I went through your protocol, In my experience adding ice cold ethanol instead of Isopropanol would be a better strategy, as ethanol helps in precipitating the DNA. Thats what we follow
Hi MIlo
I would suggest you to incubate the sample a bit longer - say at 37C overnight (shaking) and then proceed with your protocol and add ice cold ethanol and then wash…i would also suggest if you have good number of sample with you you should do two simultaneous experiment - one with ethanol and one with isopropanol…i think it should not give you any problem..but just to get confirmed you can run this...
I also use CTAB extraction for tree DNA extraction, and am following the Doyle protocol: after chloroform extraction they suggest to add just one volume of cold isopropanol (not salt+ethanol). With plants this protocol works fine, in addition I remember that I used isopropanol even with other animal tissue DNA extraction. If you want to use salt then add 2.5 volumes of ethanol, but be careful with final salt concentration. Finally, sometimes you don't see the precipitate because it is a small amount: you can leave the tube ovenight at -20 and then centrigìfuge for at east 15-20' at a speed of 5000 rpm or more (if you don't need high molecular weight DNA). cheers
- Badges
- Science method