Standard taq reaction buffer

Relative quantification of the 18S rRNA gene of M. hapla inside tomato roots infested with J2 with and without attached K6 strain were determined using 5 µl of cDNA samples. A 750 bp fragment of the M. hapla 18S rRNA of invaded J2 in roots was amplified in real-time qPCR using a CFX Connect Real Time Detection System (Bio-Rad) in 25 µl reactions containing Standard Taq Reaction Buffer (New England BioLabs), 0.25 mM MgCl₂, 0.2 mM of each dNTP, EvaGreen (Biotium), 1 µM of Meloidogyne spp. specific primers Melo1f (5′-AAGATATCTGGTTGATCCTGCCTGA-3′) and Melo723r (5′-GTTCAAAGTAAACTTGCAAYGMCTG-3′), 0.625 U Hot Start Taq DNA Polymerase (New England BioLabs). After initial denaturation at 94°C for 2 min, fragments were amplified in 40 cycles with a denaturation at 94°C for 20 s, annealing at 57°C for 20 s, extension at 72°C for 45 s, and fluorescence detection at 80°C for 30 s. Melting curves were generated between 65° and 95°C. Ct values were normalized to the ubiquitin gene expression, and 18S rRNA of J2 with attached bacterial strain K6 were determined relative to 18S rRNA of J2 without K6 using the 2^–ΔΔCt method (Pfaffl, 2001 (link)).

Four SNPs were genotyped in TLR genes: TLR1 1805G>T (rs5743618), TLR2 T>C (rs1816702), TLR2 T>C (rs4696483), and TLR4 A>G (rs1927911) by the PCR-SSP (polymerase chain reaction-sequence-specific primer) technique. The primers were designed and optimized at the Institute of Immunology, in the Faculty of Medicine–Coimbra University, Portugal (Table 1), and instructions were followed from TLR box 1.0tbox10, Instructions manual, Biocant (2010) (Biocant 2010 ). The amplification was standardized in a final volume of 15 μl: 150–300 nmol of each primer, 10 ng of DNA, 1x Standard Taq Reaction Buffer (New England Biolabs, United Kingdom), 4 mmol MgCl₂, 3% glycerol, 0.15 mg of cresol red, 100 µmol of each dNTP, and 0.1 Unit of Taq DNA Polymerase (New England Biolabs, United Kingdom) in a thermocycler (Applied Biosystems, Foster City, CA, United States), using the following parameters: one cycle: 96°C, 1 min; five cycles: 96°C, 25 s, 70°C, 45 s, 72°C, 30 s; 21 cycles: 96°C, 25 s, 65°C, 45 s, 72°C, 30 s; four cycles: 96°C, 25 s, 55°C, 1 min, 72°C, 1 min; 72°C, 10 min; hold at 4°C (optional). Next, the amplified products were separated on 2% agarose gel electrophoresis and compared by molecular sizes, after gel staining with SYBR^® Safe (Life Technologies^®, United States). Positive reactions were observed under UV light and photo-documented.

All mouse protocols were carried out in accordance with the standards of the UConn Health Animal Care and Use Committee (AP-200074-0623). The generation of mice carrying a targeted disruption of exon 1 of Gnas (Gnas E1+/-) was described previously [22 (link)]. Mice were maintained on a pure 129SvEv background and were genotyped by PCR analysis. Each 20 μL reaction was performed with 2 μL DNA, 3μL of 10x Standard Taq Reaction Buffer (NEB), 0.1 μL Taq Polymerase (NEB), 0.2ul of 40μM Gnas Forward and Gnas Reverse, 0.3ul of 40uM Neo1 primers (sequences below), 1ul of 10mM of dNTP mix (Promega), and 13.1 μL of InVitrogen Ultrapure Distilled Water. Reaction parameters were carried out as follows: 95°C for 5 minutes, 34 cycles of: (1) 95°C for 30 seconds; (2) 60°C for 30 seconds; (3) 72°C for 30 seconds, and 72°C for 5 minutes. All mice that carry a mutant maternal Gnas allele are hereafter referred to as Gnas E1+/-m and those with a mutant paternal allele as Gnas E1+/-p. Wild type mice (Gnas E1+/+) are referred to as WT.