Our GenElute™ Blood Genomic DNA Kit provides a simple and convenient way to isolate pure genomic DNA from fresh or aged (older than 24 hours) whole blood. The kit combines the advantages of silica binding with a microspin format, and eliminates the need for expensive resins, alcohol precipitation, and hazardous organic compounds such as phenol and chloroform. The starting material is lysed in a chaotropic salt-containing solution to insure the thorough denaturation of macromolecules. The addition of ethanol causes the DNA to bind when the lysate is spun through a silica membrane in a microcentrifuge tube. A Prewash Solution is provided to help remove contaminants that are associated with aged (older than 24 hours) whole blood samples. After washing to remove contaminants, the DNA is eluted in 200 µL of a Tris-EDTA solution.
The expected yields of genomic DNA will vary depending on the amount and nature of the starting material used (for example, 4–10 µg of RNase A-treated DNA can be isolated from 200 µL of fresh whole blood in less than one hour). DNA purified with this kit has an A260/A280 ratio between 1.6 and 1.9 and can be up to 50 kb in length. This DNA is ready for downstream applications such as restriction endonuclease digestions, PCR, Southern blots, and sequencing reactions.
*Note: To ensure proper fit of all tubes, a 24-place rotor is recommended. If you are using a 36-place rotor, we recommend using every other place for proper tube fit
Store the kit at room temperature. If any kit reagent forms a precipitate, warm at 55–65 °C until the precipitate dissolves and allow to cool to room temperature before use.
See Appendix 1 convert g-force to RPM
Note: If minimally sheared genomic DNA is desired in downstream applications, e.g., if using the end product for long amplification PCR, mix with gentle pipetting or inversion until homogeneous instead of vortexing in the procedure that follows.
The GenElute™ Blood Genomic DNA Kit is designed so that the DNA always remains in solution, which avoids resuspension issues. However, if it is necessary to concentrate the DNA, ethanol precipitation in the presence of sodium acetate is recommended.1
The concentration and quality of the genomic DNA can be determined by spectrophotometric analysis and agarose gel electrophoresis. Dilute the DNA in TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0–8.5) and measure the absorbance at 260 nm, 280 nm, and 320 nm using a quartz microcuvette. The absorbance at 260 nm should be between 0.1 and 1.0 (or within the linear range of your spectrophotometer). The 320 nm absorbance is used to correct for background absorbance. An absorbance of 1.0 at 260 nm corresponds to approximately 50 µg/mL of doublestranded DNA. The A260–A320/A280–A320 ratio should be 1.6–1.9.
The size and quality of the DNA can be determined by agarose gel electrophoresis.1 A gel containing 0.8% agarose (A9539) in 0.55 TBE Buffer (T6400) works well for the resolution of genomic DNA. The DNA can be visualized by staining with an intercalating dye such as ethidium bromide (E1510) and measured against a known DNA marker such as Lambda DNA Hind III digest (D9780). The genomic DNA should migrate as a single, high molecular weight band with very little evidence of shearing. A more precise determination of the size of the DNA can be made by pulsed-field gel electrophoresis.2
Figure 1. Genomic DNA purified by GenElute™ Blood Genomic DNA kits is suitable for restriction enzyme digestions.
Restriction Enzymes, EcoR I and Hind III were used to digest genomic DNA isolated with GenElute™ Blood Genomic DNA kit. Whole blood was collected in 3 different anticoagulants: EDTA, heparin, and sodium citrate. A 100 ng aliquot of genomic DNA from each anticoagulant was initially digested with EcoR I (5 units per 1 μL digested at 37 °C for 1.5 hours) and Hind III (10 units per 1 μL digested at 37 °C for 1.5 hours) followed by electrophoresis (50 ng/lane) on a 0.8% agarose gel. Ladder (M) used was Lambda Hind III (Prod. No. D9780).
Note: All centrifugation speeds are given in units of g. Please refer to Table 2 for information on converting g-force to rpm. If centrifuges/rotors for the required g-forces are not available, use the maximum g-force possible and increase the spin time proportionally. Spin until all liquid passes through the column.
See table above for spin speeds in RPM for selected common centrifuges and rotors. The correct RPM for unlisted rotorscan be calculated using the formula:
RPM = √[RCF/(r × 1.118)] × 1 × 105
where RCF = required gravitational acceleration (relative centrifugal force) in units of g;
r = radius of the rotor in cm;
rpm = the number of revolutions per minute required to achieve the necessary g-force
Up to 500 µL of blood can be used with this kit. Reagents must be increased accordingly. (Lysis Solution C and ethanol additions should be approximately 50 µL over the volume of whole blood added in the preparation.) This will decrease the number of preparations you can get from the kit. The binding column will have to be filled and spun several times to load all of the lysate from step 5, depending upon the volume used. For example, add 50 µL of Proteinase K to a 2 mL tube, 500 µL of whole blood, 40 µL of RNase A, and 550 µL of Lysis Solution C. Mix and incubate at 55 °C for 10 minutes. Add 550 µL of 95–100% ethanol and mix. Load onto the binding column (3 x 600 µL) and spin as in step 6. Continue with steps 7–9 as noted in the Procedure.
The GenElute™ Blood Genomic DNA Kit is for laboratory use only, not for drug, household, or other uses.
The Lysis Solution C contains a chaotropic salt, which is an irritant. The Column Preparation Solution is an irritant. Avoid contact with skin. Wear gloves, safety glasses, and suitable protective clothing when handling these solutions or any reagent provided with the kit. Please consult the Safety Data Sheet (SDS) for information regarding hazards and safe handling practices.