Isolate 2 plant dna kit

Caecal DNA was extracted following the protocol of ISOLATE II Plant DNA Kit (Bioline, NSW, Australia) with slightly modification. Approximately 200 mg of freshly defrosted caecal content were placed in a 2-mL Eppendorf tube contained 300 mg of glass beads. An aliquot of 450 μL Lysis Buffer PA1 was added to the samples and thoroughly mixed with a vortex mixer. The samples were transferred to a block bead mill (Retsch GmbH & Co, Haan, Germany) to disrupt at a frequency of 30/s for 5 min prior to being heated at 95 °C for 5 min. The digesta was lysed and homogenised after adding 200 μL and then 100 μL of Extraction Buffer with vortex-mixing following each addition. An aliquot of 10 μL of RNase was added to 600 μL of the lysate in a 1.5-mL microcentrifuge tube in order to remove RNA. The solution was incubated at 65 °C for 10 min. The incubated mixture was centrifuged for 1 min at 11,000 × g to pellet potential impurities. An aliquot of 450 μL of Binding Buffer was used to capture DNA by vortexing thoroughly and then centrifuging for 1 min at 11,000 × g. Then 400 and 700 μL of Wash Buffer PAW1 and PAW2 respectively were added at independent steps to purify DNA, centrifuged for 1 min at 11,000 × g to remove the wash buffer and to dry the silica membrane completely. An aliquot of 50 μL of Elution Buffer was used to elute DNA into a 1.5-mL Eppendorf tube.