Nanodrop

Mice from different groups were separated for feces collection. The total genomic DNA from each fecal sample (200 mg) was extracted using a TIAamp DNA stool mini-kit according to the manufacturer's instructions, resuspended in 40 ml of TE buffer (pH 8.0) and quantified with an Eppendorf Nanodrop. These samples were analyzed with the qPCR assay (primers used were listed in Table S1), and the Ct values were used to calculate the proportion of bacterial taxa in the feces as described previously (Yang et al., 2015 (link)).

The Oomycetes genomic DNA was isolated following the protocol of the Sarkosyl method (Sambrook and Russel, 2001). The quality of extracted DNA was checked in 1% agarose gel and quantified using Nanodrop (Eppendorf, Hamburg, Germany). The ITS1 primer including forward and reverse primers were used for amplification- and confirmation-specific site of A. invadans isolate located in the ITS1 region using thermal cycler Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA, USA) (Table 1). The PCR mixture consisted of 25 pM of each primer, deoxynucleoside triphosphate at the concentration of 2.5 mM, 0.5 units of Taq DNA polymerase and 20 ng of A. invadans genomic DNA for a total of 50 µL reaction volume to the following cyclic condition: 95 °C for 2 min, 35 cycles of 30 s at 95 °C each, 56 °C for 45 s, 72 °C for 2.5 min and 72 °C at 5 min for the final extension. The agarose gel electrophoresis analyzes the PCR product and the target product is 234 bp (Figure 2) (Vandersea et al. 2006). The PCR products are sequenced in forward and reverse direction and contig was prepared by aligning forward and reverse sequences using DNA baser 7.0.0. Afterward, the sequence was compared with available sequences in species of Oomycetes and fungal family in the NCBI GenBank with BLAST (http://blast.ncbi.nlm.nih.gov) accessed on 14 June 2022.

Total genomic DNA was extracted from 1.0 g leaf material following CTAB method³⁵ and then purified by using the Thermo Scientific GeneJET Genomic DNA Purification Kit (# K0722). DNA quality was examined by electrophoresis in 1% agarose and quantified with a Nanodrop (Eppendorf, USA). The purified genomic DNA was stored at − 20 °C for PCR amplification.

nDNA was isolated from liver tissues of mice used for immunotoxicity studies, as per the manufacturer's protocol (Sigma, USA). The quantity and quality of the isolated nDNA were estimated (nanodrop, Eppendorf, Germany). nDNA isolated from in vivo experimental groups was amplified using mouse β actin specific forward primer (f-GCGTGGGGACAGCCGCATCTT) and reverse primer (r-ATCGGCAGAAGGGGCGGAGA) (Eurogentec, Belgium, Accession number HQ675031.1) at a concentration of 100 ng/μL per reaction mixture. PCR of nDNA was carried out as per standard conditions [30 (link)] in Eppendorf master cycler; Germany. Purity and integrity of the amplified products were checked by purity factor (260/280 nm) and agarose gel electrophoresis.

DNA was labeled using PCR. PCR template was human Alu repeat material cloned in pUC19, this repeat encompassed the start and the end of tandemly repeated AluJ and AluY (NCBI: AC002400.1, 53494–53767). Standard M13 primers were used for amplification. PCR purification was done by phenol-chloroform extraction followed by ethanol precipitation using ammonium acetate as a salt. DNA concentration and incorporation of dUTP-TAMRA were measured using Nanodrop (Eppendorf) and calculated by comparing the signal before and after PCR re-precipitation.

Total genomic DNA (gDNA) was isolated from the fresh leaf tissues of the two Eryngium genotypes using a plant gDNA extraction kit (GSure^® Plant Mini Kit with WLN Buffer, GCC Biotech Pvt Ltd., Kolkata, India) following the manufacturer’s instructions. The gDNAs were quantified using a nanodrop (Eppendorf, Hamburg, Germany) and checked on 0.8% agarose gel electrophoresis (Tarson, Kolkata, India). The gDNA concentration was adjusted to 50 ng µL⁻¹, and was used for PCR amplification with the DNA barcode primers, matK, Kim matK, rbcL, and ITS, as recommended by the Consortium of Barcode of Life–Plant Group [14 (link)]. The details of the barcode primers are presented in Table 1.