The Easy-DNA Kit from Invitrogen allows for quick and easy genomic DNA isolation from many different sources. This includes blood samples, tissue culture cells, mammalian tissue, E. coli, yeast cells, plant leaves, mouse tails, and baculovirus particles. The amount of DNA that can be obtained from these different sources varies, but according to Invitrogen, 5-10 ng of DNA can be obtained from as little as 1ul of blood. And 1 cm of mouse tail will yield 125 ug of DNA. The kit comes with two proprietary solutions (solution A and solution B), TE buffer, mussel glycogen, RNAse A, and a Protein Degrader. The protocol is fairly straight-forward - cells are lysed with solution A at 65
oC, followed by precipitation and extraction of the proteins and lipids with solution B and chloroform. The mixture is centrifuged and the DNA in the upper, aqueous phase is removed and recovered by ethanol precipitation. The DNA is then suspended in TE buffer for downstream applications.
In our laboratory, we follow the protocol for DNA isolation from yeast cells and we use both small-scale (2-5 ml culture) and large-scale (>10 ml culture) procedures. For DNA isolation from Histoplasma capsulatum yeast cells, cultures are grown in 5 ml or 50 ml HMM to a cell density between 1x108 to 5x108 cells per ml. For strains containing alpha (1-3) glucan in the cell wall, cultures are grown in HMM supplemented with 0.2% 2-deoxyglucose. The alpha (1-3) glucan polymer is resistant to zymolase treatment and is alkaline soluble. Cells are washed 3 times in SCED buffer and resusupended in 200 ul (or 2ml for the large-scale protocol) SCED at 37oC for 1 hour. Cells are dispersed evenly in 350 ul (3.5ml) lysis solution A by vortexing and incubated at 65 oC for 10 minutes. Precipitation solution B is added, 150 ul (1.5 ml), to the sample and vortexed until the mixture is homogenous. Extraction with 500 ul (5 ml) of chloroform followed by centrifugation separates the DNA into the aqueous phase.
DNA is precipitated with two volumes of 100% ethanol and can either be centrifuged or spooled. For high molecular weight DNA with limited shearing, we like to spool the DNA onto sterilized pasteur pipettes, rinse in 70% ethanol, air dry, and resuspend in 200 ul (2 ml) TE buffer overnight. The sample is treated with RNAse A at 40 ug/ml at 37 °C for 30 minutes.
Typical DNA yield from a 5 ml culture ranges from 3 to 7 ug of total DNA, and from a 50 ml culture between 150 to 300 ug total DNA. The typical A260/A280 ratios tend to be between 1.2 to 1.5 with or without phenol/chloroform extractions. However, the DNA is adequate for PCR analysis and Southern blots.
The cost of the Invitrogen Easy-DNA kit is not cheap, but its advantages are ease-of-use and speed. In my mind, this makes it worthwhile.
Vincent Magrini, Ph.D.
Department of Molecular Microbiology
Washington University School of Medicine, St. Louis