The QIAamp DNA Blood Mini Kit (Blood and Body Fluid Spin Protocol) is used to provide a fast and easy method for purification of total genomic DNA for reliable real time, quantitative PCR (qPCR) reactions. The purified DNA is free of proteins, nucleases and other contaminants and inhibitors. DNA purified using QIAamp kit is up to 50 kb in size, with fragments of approximately 20-30 kb predominating. DNA of this length denatures completely during thermal cycling and can be amplified with high efficiency.
I use this system to purify DNA from frozen blood in order to perform real time qPCR on the Bio-Rad’s iQ Real-Time PCR System. This QIAamp procedure is suitable for use with either fresh or frozen whole blood and blood which has been treated with citrate, heparin, or EDTA. DNA is first absorbed onto the QIAamp silica-gel membrane during a brief centrifugation. Salt and pH conditions in the lysate ensure that protein and other contaminants which can inhibit PCR and other downstream enzymatic reactions, are not retained on the QIAamp membrane. The lysate buffering conditions are adjusted to allow optimal binding of the DNA to the QIAamp membrane before the sample is loaded onto the QIAamp spin column. DNA bound to the QIAamp membrane is washed in two centrifugation steps. The use of two different wash buffers significantly improves the purity of the eluted DNA. The wash buffers ensure complete removal of any residual contaminants without affecting the DNA binding. I elute the purified DNA from the QIAamp spin column in a concentrated form using water, though elution buffer is provided. The purified DNA can more readily be used for different applications if eluted in distilled water.
There are two drawbacks to this kit. The kit does not include enough 2 ml collection tubes in order to carryout the additional spin cycle to eliminate any chance of possible wash buffer carryover before the final elution. The kit does not come with enough reagents so make sure to order additional Proteinase K and Buffer AL.
For the final elution, it is recommended to incubate the QIAamp Sin Column loaded with distilled water for 10 minutes at 65ºC in order to increase DNA yield; this is not mentioned in the protocol manual.
One other blood purification kit that I’ve tried before is the Wizard Genomic DNA purification kit. In comparison, the QIAamp kit is easier to use and provides higher DNA yields.