Ready to go pcr beads

Two regions of the genomic island were targeted for PCR amplification. A 923 bp product at the 5′ end of the island was amplified using BF1 5′-ATCTTTTCCGCGAATCACTG-3′ (Venter 70585 2049624-2049643) and BR1 5′-TTGTATTGGAGGACCAAGC-3′ (2050519-2050537) primers. Also a 1323 bp product was amplified targeting the 3′ end with EF1 5′-GCGCTGTAATATAGGCAAAGC-3′ (2054079-2054099) and ER1 5′-ATAAGCGTGTCCGCTATCGT-3′ (2055382-2055401) primers. Bacterial colonies were inoculated into each PCR reaction consisting of 12.5 μL of HotStart Taq mastermix (Qiagen), 1.25 μL of each 10 μM primer, and 10 μL of ddH₂O. Reactions were incubated at 95°C for 15 minutes, followed by 30 cycles each of 94°C for 30 s, x°C for 30 s, and 72°C for 60 s, and finally 72°C for 10 min, where x equals 52.5°C for BF1/BR1 and 54.0°C for EF1/ER1 primers. Additional reactions were also carried out with Ready-To-Go PCR beads (GE Healthcare) with 0.5 μL of each 10 μM primer and 24 μL of ddH₂O. Reactions were carried out with the same conditions for each respective primer pair. Any samples with discrepant or negative PCR results were repeated.

Apothecia were cleaned with acetone before DNA extraction. DNA was extracted using the GeneOn Plant DNA Extraction Kit (GeneOn BioTech, China) by the magnetic bead method or the Chelex® 100 Resin (Bio-Rad, USA) method following Ferencová et al. (2017) (link). The fungal internal transcribed spacer (ITS) region of the rDNA was amplified via polymerase chain reaction (PCR) using the primers ITS1F (Gardes and Bruns 1993 (link)) and ITS4 (White et al. 1990 (link)). The large subunit of the nuclear ribosomal DNA (LSU) was amplified using the primers AL2R (Mangold et al. 2008 ) and LR6 (Vilgalys and Hester 1990 (link)) and the mitochondrial small subunit (mtSSU) of ribosomal RNA using the primers 16F and 972R (Li et al. 2023 (link)). PCR amplifications were performed in 25 µl volumes Ready-To-Go PCR Beads (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) containing 5 µl of DNA extract and 1 µl of each primer. Cycling conditions included initial denaturation at 94 °C for 5 min, followed by 4 cycles of 94 °C for 30 s, 54 °C (53 °C for mtSSU) for 45 s and 72 °C for 60 s, 30 cycles of 94 °C for 30 s, 48 °C for 30 s and 72 °C for 60 s and a final extension at 72 °C for 10 min. The PCR products were visualised on 1% agarose gels and sequenced by Macrogen Europe (Amsterdam, The Netherlands) with the same primers as the original PCR amplifications.

PCR analysis and partial sequencing of 7 housekeeping genes was performed on 86 out of 87 ORT strains. Gene sequences from the ORT type strain DSM 15997 were taken directly from GenBank (http://www.ncbi.nlm.nih.gov/genbank/index.html).
The primer sets used for MLST analysis (Table 2) were created based on the genomic sequence of ORT type strain DSM 15997 using the primer designing tool Primer 3 [28 ]. For the ORT reference strain of serotype F (RefF), a different reverse primer (5’-TCRTTCCATTTRTTTTGTCCTT-3’) was used for PCR amplification and partial sequencing of the housekeeping gene adk to yield the desired amplicon.
PCR was carried out using Ready-To-Go PCR beads (GE Healthcare, Freiburg, Germany) with 25 μl reaction volumes containing 1 μl of bacterial DNA and 1 μl of the respective forward and reverse primers (25 pmol/μl). PCR cycler conditions for all genes were the following: initial denaturation at 94°C for 5 minutes followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 52°C for 60 seconds and extension at 72°C for 90 seconds. The final extension at 72°C for 7 minutes completed the PCR. Clean-up of PCR products and Sanger sequencing in both directions were performed by microtitre plate sequencing at LGC Genomics, Berlin, Germany.

DNA from whole mosquitoes was prepared with the Promega Wizard Genomic DNA Purification Kit according to the manufacturer’s instructions. Bacterial 16 S rDNA (Escherichia coli position 341–805) were amplified by using general bacterial primers 341F (CCTACGGGNGGCWGCAG) and 805R (GACTACHVGGGTATCTAATCC)26 (link). This primer pair matches approximately 90% of all good-quality bacterial sequences and covers all phyla in the Ribosomal Database Project release 10.25. Each DNA sample was individually PCR-amplified with Ready to go PCR beads (GE Health Care) by initial denaturation at 95 °C for 5 min followed by 35 cycles of [40 s at 95 °C, 40 s at 53 °C and 1 min at 72 °C] followed by a final 7-min extension at 72 °C. In a second PCR was added 1 of 50 flanking barcode sequence pairs to run samples in parallel27 (link) using the same conditions as above, but only for 10 cycles of iteration.

The transcriptomic analysis has been carried out using RT² Profiler PCR Array, a commercially available 96-well plate using real-time PCR (RT-qPCR) approach. In brief, HepG2 cells were seeded in 6-wells plate and treated with 100 µM of TEHP for 72 h. Total RNA was isolated by using RNeasy Mini Kit (Qiagen, Germantown, MD, USA). Purity of RNA was measured on Nanodrop 8000 and cDNA synthesis was carried out using ready-to-go PCR beads (GE Healthcare, Chalfont Saint Giles, UK). Transcriptomic changes of 84 genes belonging to human cancer pathway was analyzed by RT² Profiler™ PCR Array (PAHS-033Z, SA Biosciences Corporation, Allentown, PA, USA) on Roche^® LightCycler^® 480 (Roche Diagnostics, Rotkreuz, Switzerland). Expressional changes in TEHP-treated cells were normalized with five housekeeping genes and the results are represented heat map, scatter plot, and fold change [29 (link)].