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Important
Below is one example of a method to extract DNA from swine blood samples. However, users can instead use other options (e.g. commercially-available DNA extraction kits) if preferred. The authors have found that spin columns are not suitable for this protocol due to PCR inhibitors being carried over in the blood. This method is optimised for frozen blood samples; other methods may work with fresh blood.
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Transfer 50 μl of whole blood to a 1.5 ml Eppendorf DNA LoBind tube.
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Add 1 ml of lysis buffer to each sample.
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Incubate on a Hula mixer (rotator mixer) for 1 minute at room temperature.
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Remove the samples from the Hula mixer and incubate at room temperature for 30 minutes.
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Centrifuge at 7,500 rpm for 5 minutes. You should see a pellet form at the bottom of the tube.
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Without disturbing the pellet, remove and discard the supernatant.
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Add 700 μl of 5X TEN buffer to each sample.
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Incubate on a Hula mixer (rotator mixer) for 1 minute at room temperature.
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Remove the samples from the Hula mixer and incubate at room temperature for 30 minutes.
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Incubate the samples at 95°C for 5 minutes in a heat block or water bath.
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Centrifuge at 7,500 rpm for 5 minutes. You should see a pellet form at the bottom of the tube.
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Without disturbing the pellet, remove and discard the supernatant.
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Resuspend each pellet in 700 μl 100% isopropanol.
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Incubate on a Hula mixer (rotator mixer) for 1 minute at room temperature.
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Remove the samples from the Hula mixer and incubate at room temperature for 30 minutes.
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Prepare sufficient fresh 70% ethanol in nuclease-free water.
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Centrifuge the samples at 14,500 rpm for 15 minutes. You should see a white pellet form at the bottom of the tube.
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Without disturbing the pellet, remove and discard the supernatant.
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Wash the pellet by adding 500 μl of freshly-prepared 70% ethanol and pipetting up and down.
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Centrifuge the samples at 11,500 rpm for 5 minutes.
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Without disturbing the pellet, remove and discard the supernatant.
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Allow the pellet to dry for ~30 seconds, ensuring there is no ethanol left in the sample. However, do not dry the pellet to the point of cracking.
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Resuspend the pellet in 30 μl nuclease-free water. If it is difficult to resuspend in this volume, add more nuclease-free water.
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End of step
The resuspended samples will be taken forward into the tiled DNA amplification and clean-up step. The extracted DNA can be used immediately or stored at 4°C for up to one week. For longer-term storage place at -20°C or -80°C.