Deoxy ribonucleoside triphosphates dntps

Terminal deoxynucleotidyl transferase (TdT), deoxy-ribonucleoside triphosphates (dNTPs), deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Klenow fragment polymerase (3′-5′exo-, KF polymerase) and the nicking endonuclease Nt.BstNBI were bought from New England Biolabs Ltd. (Beverly, MA, USA). Tris [Tris-(hydroxy-methyl) aminomethane], hydrochloric acid (HCl), thioflavin T (ThT), sodium chloride (NaCl), magnesium chloride (MgCl₂), were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). DNA and RNA sequences were synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). All other reagents were of analytical reagent grades. Ultrapure water (18.2 MΩ.cm) used in the experiments was obtained from a Milli-Q water purification system (Millipore Corp, Bedford, MA, USA). The DNA sequences and RNA sequence list were as follows: miRNA-21, 5′-UAG CUU AUC AGA CUG AUG UUG A-3′; cDNA: ATA TCA GCG ATC ACC CAT GTT ACT CTC TAA CAG ACT CTC AAC ATC AGT CTG ATA AGC TA-3′. The reaction buffer used in this study contained two components: (1) Tris-HCl buffer (50 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl₂, pH 7.9), (2) 1 × TdT buffer.

A comparison of the caprine KRTAP15-1 sequence (accession number
AY510116.1) and the ovine KRTAP15-1 sequences (accession numbers
MH742372 – MH742375) suggested that the primers designed for ovine
KRTAP15-1 (Li et al., 2018), would also amplify a 495 bp fragment
covering the entire coding sequence of caprine KRTAP15-1. The
sequences of these primers were 5'-GAACTCAGAACTCCCAACAG-3' and
5'-TAACCATGAGGTGACTGGAG-3', and they were synthesised by Sangon Biotech Co.,
Ltd (Shanghai, China).
Amplifications were undertaken in Bio-Rad S1000 thermal cyclers (Bio-Rad,
Hercules, CA, USA) and performed in a 20  $\mathrm{\mu }$ L reaction including the
purified genomic DNA from one 1.2 mm punch of dried blood, 0.25  $\mathrm{\mu }$ M
of each primer, 2.0  $\mathrm{\mu }$ L of $\mathrm{10}\times$  PCR buffer
(supplied with the DNA
polymerase enzyme), deoxyribonucleoside triphosphates (dNTPs) at
150  $\mathrm{\mu }$ M (Takara, Dalian, China), 2.5 mM Mg ${}^{\mathrm{2}+}$ and 0.5 U of
Taq DNA polymerase (Takara), with deionised water ( ${\mathrm{dH}}_{\mathrm{2}}\mathrm{O}$ ) to make
up the volume. The PCR amplification conditions consisted of an initial
denaturation at 94  ${}^{\circ }$ C for 5 min, followed by 35 cycles of
94  ${}^{\circ }$ C denaturation for 30 s, 60  ${}^{\circ }$ C annealing for 30 s and
72  ${}^{\circ }$ C extension for 30 s, and a final extension at 72  ${}^{\circ }$ C
of 5 min. The quality of PCR products was examined using agarose gel
electrophoresis (1  % gel in $\mathrm{1}\times$  TBE (Tris–borate–EDTA) buffer).

All reagents and chemicals were of the highest quality for analytical application. The American Type Culture Collection (ATCC) was consulted for strains of Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Bacillus coagulans (B. coagulans), and methicillin-susceptible Staphylococcus aureus. Thermo Scientific (USA) supplied the Bsm DNA polymerase and the Nb. Bpu10I nicking endonuclease. Deoxyribonucleoside triphosphates (dNTPs) were procured from TaKaRa Bio Inc. (China). The DNA sequences listed in Table S1 were synthesized and HPLC-purified by Sangon Biotech Co., Ltd. (China).