DNeasy® Blood and Tissue Kits provide all reagents and most of the tubes needed for rapid purification of total genomic DNA from a variety of different tissues, cells, blood and bacteria. The kit is available as individual columns (50 or 250 columns) or as 96-well plates (4 or 12 plates) for high-throughput purification. Purification of DNA with the DNeasy® kit is based on binding genomic DNA to a silica membrane, thereby avoiding the use of extractions with hazardous organic solvents like phenol or chloroform. In addition, there is no DNA precipitation with ethanol necessary. This makes the kit highly suitable for processing multiple samples at the same time, e.g. for isolation of tail DNA when screening a transgenic mouse colony. After enzymatic tissue lysis with proteinase K overnight at 56°C, the DNA is bound to the silica within a DNeasy mini spin column (or well of a 96-well plate) while contaminants, proteins and divalent cations are removed during centrifugation. The DNA bound to the column is washed twice to further remove contaminants and enzyme inhibitors and then eluted either with low-salt buffer or dH2
O depending on the downstream application(s). The DNA can be used in applications like PCR, Southern blot, AFLP and RFLP. The predominant fragment size of the purified DNA is 30 kb, but can be up to 50 kb.
I used the DNeasy® Kit in the spin-column format to isolate genomic DNA from tails of mice from different lines for screening by PCR; purified DNA was also used for enzymatic digestion followed by ligation of linker DNA. The procedure is easy to follow using the well-written protocol and all solutions are provided. Since I used the DNA in enzymatic digestions and subsequent ligations, I added the recommended step of digesting the RNA by incubating the lysed tails with 4 µl of a 100 mg/ml RNase A solution (not provided in the kit). Using a 0.6 cm mouse tail for DNA purification, Qiagen estimates a yield of 10-25 µg DNA, but I found that the concentrations of the eluted DNA varied over a wider range between animals (when isolating wildtype and transgenic DNA) as I achieved yields from 6 to 43 µg. Since I was using the purified genomic DNA in restriction digests, I preferred to elute with dH2O or 10 mM Tris/HCl, pH 9.0 instead of the EDTA-containing elution buffer (10 mM Tris/HCl, 0.5 mM EDTA pH 9.0).
PCR reactions always worked with the purified DNA and sometimes my digestions worked as well. The fact that some of my digestions were incomplete or did not work at all may be due to the fact that some enzymes are more sensitive to impurities in the DNA or perhaps the restriction site still had proteins bound to it. For these problematic digests, Qiagen’s technical service suggested that I re-purify the DNA in order to remove possible DNA-bound proteins or contaminants. They recommended that I first add 1/10 of the first washing buffer (AW1) and 2.5 times of the second washing buffer (AW2) to the DNA, incubate the mixture for 3 min and purify the DNA with the spin column again, according to the protocol. The amount of re-purified DNA was low but pure enough to yield the expected digestion pattern after restriction digestion. Qiagen’s technical support was excellent.
The DNeasy® Kit is a reliable and easy to follow method for the isolation of genomic DNA from mouse tail, especially for lab personal with little experience in molecular biology. It is a fast procedure even when handling multiple samples for screening a transgenic colony. After the initial overnight proteinase K digestion, it takes about 1 h to purify 18 samples simultaneously. The yield and purity of DNA eluted from the column is sufficient for screening with PCR and most downstream applications, but purity can be improved with additional purification steps.
DNeasy Blood And Tissue Kit From Qiagen
Fast, easy and reproducible purification of genomic DNA from a variety of sources without the use of phenol or chloroform. Especially useful for high-throughput purification.
DNA fragmentation into 30-50 kb interferes with some downstream applications where intact DNA is required. Lots of plastic waste.
The Bottom Line
Excellent kit; contains all necessary reagents for purification of genomic DNA for most downstream applications in an easy to follow and rapid procedure, even when used for high-throughput.