Hi, I am performing genomic DNA extractions from protozoa cell pellets using a phenol chloroform extraction + EtOh precipitation protocol. Although I am being able to get a decent amount of DNA (1-2ug in 50ul of TrisHCl), my A260/280 ratios are really low (1-1.5). I tried to troubleshoot it by performing a second precipitation using 2.5 volumes of ice cold EtOH and 1/10 volumes of Na acetate. However, my DNA recovery after this step is less than 30% with no improvement of the purity ratio at all. Has anyone been trough something similar? Any suggestions would be highly appreciated!
Hi, I will recommend you to use Phenol:chloroform: Isoamyl alcohol (25:24:1) instead of Phenol: chloroform, the interphase of isoamyl alcohol will help in pipetting out the aqueous phase without contamination. For precipitation use 100% Isopropanol (chilled) which will increase total DNA content but may result in precipitation of salts which can be removed by washing the pellet with 75% Ethanol and then resuspend it in suitable buffer/water.
Use 90% ethanol for precipitation and shake well. Further store it at -20 deg C for overnight. It helps in better yield.