Qiaquick nucleotide removal kit

Plasmid DNA was linearized with appropriate restriction endonucleases for 5 h at 37 °C, purified using QIAquick Nucleotide Removal Kit (Qiagen, Hilden, Germany), and the degree of linearization was examined on a 1% agarose gel. In vitro transcription to produce digoxigenin (DIG)-labeled RNA probe was carried out combining linearized plasmid, 1 μg DIG labeling mix (Roche, Mannheim, Germany), 2 μl transcription buffer, 2 μl RNase inhibitor (Roche, Mannheim, Germany), 1 μl T7/Sp6 RNA polymerase (Roche), and 2 μl RNase-free ddH₂O to a final volume of 20 μl. The mix was incubated at 37 °C for 2 h. This was followed by DNase I treatment for 15 min at 37 °C. Labeled RNA was purified using the RNeasy Mini Kit (Qiagen, Hilden, Germany); probe length was verified by agarose gel and then dissolved in 150 μl hybridization buffer and stored at − 20 °C until use.

Cytosol was isolated using the cell mitochondria isolation kit (C3601, Beyotime Institute of Biotechnology, Nantong, Jiangsu) according to the manufacturer’s instructions as previously described [37 (link), 43 (link)]. In brief, 1 × 10⁷ cells were incubated in 0.1 ml ice-cold mitochondrial lyses buffer for 10 min and homogenized with a Dounce homogenizer for 30 strokes. The homogenate was centrifuged at 600 × g for 10 min at 4 °C to remove nuclei and unbroken cells. The supernatant was collected and centrifuged again at 12,000 × g for 30 min at 4 °C for production of a supernatant corresponding to the cytosolic fraction. DNA of cytosolic fractions were isolated using QIAQuick nucleotide removal kit (28306, QIAGEN, Valencia, CA) following the manufacturer’s protocol. The copy number of mtDNA was measured by qPCR with same volume of the DNA solution as previously described [44 (link)].

Genomic DNA from the virulent LACK/LACKL. major control line as well as the LACK/LACK^RDG and LACK/LACK^DDE lines was extracted with phenol-chloroform (34 (link)), digested overnight with StuI, concentrated by ethanol precipitation, and size separated by agarose gel electrophoresis. The DNA was transferred onto Hybond-N+ nylon membrane (GE Healthcare Life Sciences, Piscataway, NJ) by alkali capillary blotting following the manufacturer’s instructions. A biotin-labeled probe, derived from the LACK ORF, was synthesized using a NEBlot Phototope kit (New England BioLabs, Ipswich, MA) and purified using a QIAquick nucleotide removal kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s recommendations. The labeled probe was hybridized to the target DNA at 65°C using Rapid-hyb buffer (GE Healthcare Life Sciences) according to the manufacturer’s instructions. Following hybridization, the membrane was washed in 2× saline sodium citrate (SSC) plus 0.5% SDS followed by 0.2× SSC plus 0.5% SDS as previously reported (7 (link)). The blot was visualized by chemiluminescence using the Phototope-Star detection kit (New England BioLabs) as per the manufacturer’s specifications.

DNA vectors were labeled using the Cy3-LabelIT reagent kit (Mirus, Madison, WI, USA). In each 50 μL reaction, 5 μg DNA was labeled with 1 μL of reagent. After one hour incubation at 37°C, unreacted reagent was removed using QiaQuick Nucleotide Removal kit (Qiagen). A NanoDrop ND-1000 Spectrophotometer (ThermoFisher Scientific) was used to measure absorbance at 260 nm and 550 nm, which was used to calculate the DNA and Cy3 concentrations, respectively.

For each decoction sample collected at different times of boiling, large sediments were removed by MiniSpin centrifuge (Eppendorf, Hamburg, Germany). One portion of the supernatant was used for direct PCR and was regarded to be “crude DNA”. Another portion was lyophilized, followed by DNA extraction using a QIAquick Nucleotide Removal kit (Qiagen, Hilden, Germany) according to manufacturer instructions for the single herb decoctions; modified cetyltrimethylammonium bromide (CTAB) DNA extraction (protocol 1) (Additional file 1) for the multi-herb decoctions; and modified CTAB DNA extraction (protocol 2) (Additional file 1) for the Korean Ginseng Chicken Stew. This portion was regarded to be “extracted DNA”. Crude and extracted DNA were stored at −20°C immediately and PCR analyses were carried out within one month.