Dntp mix

Genomic DNA of each plant from SI line S-1300, 11 SC accessions, 11 SI F₁ hybrids and 4552 individuals of F₂ and BC₁ populations, was isolated from young leaves using cetyltrimethyl ammonium bromide [44 ]. DNA concentration was measured using a Beckman spectrophotometer. DNA from three individuals in each of the SI line S-1300, 11 SC accessions and 11 SI F₁ hybrids was mixed for PCR analysis. PCR was performed on a thermocycler (Model PTC-225, MJ Research) in a volume of 20 μL including 50 ng DNA template, 0.2 mM dNTP mix (Sangon, China), 0.5 μM of each primer, 1 U Taq DNA polymerase (MBI Fermentas, USA), 2.0 mM MgCl₂, and 2 μL 10× Taq buffer. The PCR mixture was covered with 20 μL mineral oil [18 (link)]. PCR products were separated on a 1.0% agarose gel in 1× TAE buffer and detected by staining with ethidium bromide.

The LAMP reaction adopts a 25 μl system: 10 × Isothermal Amplification Buffer 2.5 μl, 6 mM MgSO₄ [New England Biolabs (Beijing) LTD., Beijing, China], 1.4 mM dNTP Mix (10 mM) Sangon Biotech Co. Ltd. (Shanghai, China), 0.2 μM each of the F3 and B3, 1.6 μM each of primer FIP and BIP, 0.4 μM each of primer LB and LF, Bst 2.0 DNA Polymerase (8,000 U/ml) [New England Biolabs (Beijing) Ltd., Beijing, China] 1 μl, DNA sample 1 μl, ddH₂O makes up 25 μl. The mixture was incubated at 65°C for 60 min. The reaction was terminated by heating at 80°C for 5 min and the products were separated by electrophoresis in 1.0% agarose gel. After the reaction product cooling, add 1 μl of 1/10 diluted original SYBR Green I (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) solution for color reaction.

Casein, Casein-Na, and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Shanghai, China). All oligonucleotides—including the forward primer 5′-biotin-GCTCAACCAGGAACGATC-3′ and reverse primer 5′-FITC-GCAGCATTTGACTACGTACCA-3′ (with expected amplified length of 112 bp); 4S Red Plus nucleic acid stain (1000×); Taq DNA polymerase (5 U/μL); 10 × PBS; streptavidin; agarose; dNTP mix (25 mM); and proteinase K solution (20 mg/mL)—were all obtained from Sangon Biotech (Shanghai, China). A S1000 Thermal Cycler PCR (Bio-Rad, Hercules, CA, USA) and the portable thermal controller (Hangzhou Ao-Min Biological Co., Ltd., Hangzhou, China) were used for the amplification. All components of the LFS, including a plastic adhesive backing, sample pad, conjugate pad, nitrocellulose membrane CN 95 and absorbent pad were ordered from the Shanghai Jie-ning Biotechnology Co., Ltd. (Shanghai, China). All solvents and other chemicals of analytical reagent grade were used without further purification.