The DNA-purification process was performed using the KingFisher® Flex, in combination with the KingFisher Cell and Tissue DNA Kit, which consists of: KingFisher Magnetic Beads, Lysis Buffer, Binding Buffer, three Wash Buffers and an Elution Buffer. The magnetic particles are characterized by a high surface area and uniform size distribution, making them suitable for high-throughput processing.
DNA was purified from different sample materials:
- 1 x 106 frozen HeLa-S3 cells
- One punch (0.2 cm diameter) mouse ear
- 0.1–0.2 cm mouse tail
- 15 mg mouse liver
- 15 mg mouse kidney
- 20 mg mouse spleen
In order to assess the best conditions for optimizing yield, different processing times for lysing the tissues were tested, and DNA was eluted in 150 µL of the Elution Buffer
Sample lysis. The DNA-purification process begins with homogenization and lysing of the sample. Efficient lysis is extremely important in order to gain high yields of quality DNA. The sample material must be effectively lysed to enable the magnetic beads to bind to the DNA in a suitable buffer. Subsequent washing steps dispose of inhibitors, and high-purity DNA is eluted into the elution buffer. Optimal lysis time differs between cell and tissue types, due to various cellular and extracellular molecular structures, for example hard tissues such as cartilage and bone.
Lysis of cells. After the addition of the 200 µL Lysis Buffer, the HeLa-S3 cells were efficiently mixed by pipetting and subsequent vortexing for 30 seconds, until the viscosity was lost. The samples were incubated for 15 minutes at 70ºC.
Lysis of tissue samples. Ear, kidney, and liver samples were lysed in 250 µL Lysis Buffer and vortexed for 15 seconds. Parallel samples were then incubated at 56ºC for 15 minutes, 1 hour, 4.5 hours, and overnight. Spleen and tail samples were only lysed overnight (due to limited availability of sample material). Following lysis, all samples were centrifuged for 5 minutes at 6000 x g to separate out the cell debris from the lysate.
The 15 minute lysis of the tissue samples occurred directly in the KingFisher Flex embedded heating block. The samples lysed for longer time periods (1 hour, 4.5 hours, and overnight) were incubated in a benchtop heating block.
Purification. Each sample type was purified using the magnetic bead technology of the KingFisher Flex. This ensures that the final DNA is of high quality and free from proteins, nucleases, and other contaminants or inhibitors.
Once the samples are cleared of any cellular debris, the lysate, Binding Buffer, and KingFisher Magnetic Beads were all transferred to the KingFisher Flex instrument. This technology is based on the use of magnetic rods, which move particles through the various phases of purification (binding, washing, and elution). By transferring the magnetic particles instead of liquids, there is no chance of contaminating residual carryover and a rapid and reproducible protocol is therefore ensured.
During the binding step, the target DNA binds to the magnetic beads, in the presence of chaotropic salts. In order to provide highly pure DNA, the wash steps effectively remove any potentially contaminating proteins or cellular debris. The purified DNA is then eluted from the magnetic beads into the Elution Buffer. The purification process within the KingFisher Flex took approximately 25 minutes per 96 samples.