Genomic DNA purification from whole blood Experimental methods
Genomic DNA purification from whole blood Experimental methods
The Wizard Genomic DNA Purification Kit for isolation of genomic DNA from whole blood (10 ml) is based on a four-step procedure. The first step of the purification procedure consists of lysis of the red blood cells, discarding of the soluble fraction, followed by lysis of the white blood cells and their nuclei. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Genomic DNA purification from whole blood Experimental methods
Materials and Instruments
Ethanol Isopropanol RNAase A Whole Blood Move I. Materials For more product details, please visit Aladdin Scientific website.
Agarose Gel Electrophoresis Centrifuge Tubes Wizard Genomic DNA Purification Kit
1. Buffers, solutions and reagents
Ethanol, 70%, room temperature
Isopropyl alcohol, room temperature
RNAase A
Dissolve RNAase A in DNA rehydration solution to a final concentration of 4 mg/ml and boil for 10 min to remove DNAase contamination. Dispense and store at -20°C.
2. Specialized equipment
Agarose gel electrophoresis equipment
50 ml sterilized centrifuge tubes
3. Other
Wizard Genomic DNA Purification Kit (Promega; includes Cell Lysate, Nucleus Lysate, Protein Precipitate, and DNA Rehydration Solution)
Molecular weight markers for agarose condensation electrophoresis
4. Cells and Tissues
Whole blood (10 ml)
II. Methods
1. Lysis of erythrocytes
(1) Add 30 ml of cell lysis solution to a 50 ml sterilized centrifuge tube.
(2) Gently shake the blood sample tube until the sample is completely mixed; then transfer the blood sample (10 ml) to the tube with cell lysis solution and invert the tube 5 or 6 times to mix.
(3) The mixture is incubated at room temperature for l0 min (during which time it is inverted and mixed 3 times) to lyse the blood erythrocytes, and centrifuged at 2000 g for l0 min at room temperature.
(4) Remove as much of the supernatant as possible without destroying the white visible sediment, approximately 1.4 ml of residual liquid will remain. If the blood sample has already coagulated then add
If the blood sample has coagulated, add 30 ml of cell lysate and mix by inverting 5 or 6 times. Repeat (3) and (4) until the sediment is almost completely white. There may be some loss of DNA from the clotted sample.
In step (4), some hemoglobin or cellular debris may be seen in the white blood cells as well. If the sediment appears to contain only hemoglobin cells, discard the supernatant and add another 30 ml of cell lysate to the cell sediment and repeat (3) and (4).
(5) Vigorously shake the tube until the leukocytes are resuspended (10-15 s).
2. Nuclear lysis and protein precipitation
(1) Add l0 ml of nuclear lysis solution to the tube containing the resuspended cells and aspirate the solution 5 or 6 times to lyse the leukocytes so that the solution becomes very viscous. If clumps of cells appear after mixing, incubate the solution at 37°C until the clumps are completely disrupted. If clumps are still visible after 1 h, add another 3 ml of nuclear lysate and repeat the incubation process.
(2) Optional: Add RNAase A 20ug/ml to the lysate, mix by inverting the tube 2?5 times, incubate at 37°C for 15 min, then cool to room temperature.
(3) Add 3.3 ml of protein precipitation solution to the lysate and shake vigorously for 10?20 s. Small protein clumps may be seen.
(4) After centrifugation at 2000 g for lOmin at room temperature, a dark brown white mass will be seen.
3. Precipitation and Rehydration of DNA
(1) Transfer the supernatant to a clean 50 ml test tube containing 10 ml isopropanol at room temperature.
(2) Gently invert the mixed solution until the white linear DNA morphs into visible chunks.
(3) Centrifuge at 2000 g for 1 min at room temperature and small pieces of white DNA will precipitate.
(4) Pour out the supernatant slowly at room temperature, add l0 ml of 70% ethanol, gently invert the tube several times to wash the DNA precipitates and the walls of the tube, and then centrifuge as in (3).
(5) Carefully aspirate the ethanol. The DNA precipitate is very loose at this point and care must be taken to avoid sucking the precipitate into the pipette.
Place the tube upside down on a clean absorbent paper and air dry the sediment for 10-15 min.
(6) Add 800ul of DNA rehydration solution and incubate at 65°C for lh to rehydrate the DNA, tapping the tube periodically to mix the solution. Another way to rehydrate DNA is to incubate the solution at room temperature or 4°C overnight.
(7) Store the DNA at 2 to 8°C. 
4. Characterization and Quantification of Isolated DNA
DNA can be quantified by measuring the absorbance of DNA at 260mn or by agarose gel electrophoresis to determine the amount of purified sample relative to a standard. If precise concentrations are required, the latter method is recommended rather than determination by spectrophotometry, as absorbance readings can be skewed by the presence of low molecular weight UV-absorbing substances in the sample (Glasel 1995). Typical DNA yields for 10 ml of whole blood are 250 to 500 ul (Table 9-1). 
The quality of DNA isolated by this method can be estimated by determining the amplification length and the ability of PCR to amplify it (Saikiet al. 1985). Pulsed-field gel electrophoresis shows that the majority of DNA fragments isolated by this method are in the range of 50 to 200 kilobases (Figure 9-2). The compatibility of the isolated DNA with PCR amplification can be demonstrated by the successful amplification of polymorphic loci from three individuals. Samples from 5 different individuals (25ug) were all amplified efficiently, with each locus producing the same type of parallel bands (Figure 9-3). 