Pcr buffer

The mutant library of the AHL lactonase MomL was constructed using error prone PCR (epPCR). The primers for epPCR are listed in Table S1. Each 100-μL epPCR reaction contained 10 μL of 10× PCR buffer (Takara, Shiga, Japan), 8 μL of dNTP mixture (2.5 mM dATP, 2.5 mM dGTP, 10 mM dCTP, and 10 mM dTTP), 1 μL of the primer momL-F (20 μM), 1 μL of the primer momL-R (20 μM), 1 μL of template plasmid from strain AHL882-5, 1 μL of Taq DNA Polymerase (Takara, 5 U/μL), appropriate metal ions, and deionized water to a final volume of 100 μL. PCR was conducted using the following conditions: denaturation at 94 °C for 10 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 60 s, and a final incubation at 72 °C for 10 min. The resulting PCR products were digested with Prime STAR^® HS DNA Polymerase (Takara) to improve the ligation efficiency. They were then further digested with DpnI (NEB, Ipswich, MA, USA) to remove template plasmids and were finally purified using a PCR product purification kit (Biomed, Beijing, China) according to the manufacturer’s instructions. Purified mutant momL genes were ligated into the linear vector pET-24a(+) via seamless cloning. Recombinant plasmids were transformed into E. coli BL21(DE3), diluted with fresh LB medium, plated on LB agar containing 25 μg/mL kanamycin, and cultured at 37 °C overnight.

Total RNA of DMSCs at P3, P12 and P21 passages was extracted using Trizol reagent (Invitrogen). Total RNA was reversed transcribed into cDNA, followed by 30 PCR cycles using an RNA PCR kit version 3.0 (Takara). RT–PCR primers of DMSC's surface markers are listed in Table 1. PCRs were operated in 25 μl volumes containing 2.5 μl of 10× PCR buffer (Takara), 16.75 μl of double-distilled H₂O, 0.25 μl of Ex-Taq (Takara), 1.0 μl each of forward and reverse primers and 1.5 μl of template cDNA. Cycling conditions contained an initial denaturation step at 94°C for 5 min, then 30 cycles at 94°Cfor 30 s, 50–60°C for 30 s and 72°C for 30 s. PCR products were visualized by electrophoresis on 2% (w/v) agarose gels.

Total RNA was extracted using a plant-specific RNA kit (Invitrogen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from RNA isolated from P. trichocarpa using M-MLV reverse transcriptase. Primers (Supplement Table 1) were designed and synthesized for defensin ORF cloning, the PCR system includes 2 µL forward and reverse primers, 2.0 µL cDNA as template, 5.0 µL 10× PCR buffer (Mg²⁺ plus), 1 µL 10 mM dNTPs, 0.5 µL rTaq DNA polymerase (Takara, Japan) and the ddH₂O was to a constant volume up to 50 µL. Also, the PCR reaction was performed as follows: 95 °C for 10 min, 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s and, finally, 72 °C for 10 min. Also the PtDef PCR product was purified (Takara, Japan) and inserted into the PEASY-T3 plasmid (TransGen Biotech, China). Subsequently, the positive clones were sequenced using the M13 primers. The nucleotide sequence, deduced amino acid sequence, and ORF of defensin from P. trichocarpa were analyzed using the ExPASy online package (http://www.expasy.org/translate/). We then calculated the molecular weight (Mw) of defensin and predicted its isoelectric point (pI) using the ExPASy pI/Mw tool (http://www.expasy.org/tools/pi_tool.html).

The screened strain was identified based on colony morphology, gram staining, and spore staining. Molecular identification was performed by amplification of 16S rDNA with bacterial universal primers 27F and 1492R. Total genomic DNA was extracted from the selected isolates by a rapid bacterial genomic DNA isolation kit (Sangon Biotech, Shanghai, China). Amplifications were performed in a 25 μL reaction mixture containing 1 μL of genomic DNA, 1 μL of each primer, 2.5 μL 10×PCR Buffer, 2 μL of dNTP, and 0.5 μL of Taq polymerase (Takara, Beijing, China). Samples were amplified by 30 cycles consisting of 94°C for 30 s, 54°C for 45 s, and 72°C for 50 s, followed by a final extension step of 10 min at 72°C. The PCR product was purified by the kit and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Acquired sequences were compared with the GenBank database by BLASTN to determine the homology of 16 s rDNA gene sequences.

Ethanol was decanted off specimens and eggs were soaked in 1.5 mL ddH₂O for 1 h. Water was then removed with a pipette, and egg genomic DNA was extracted using an easy-DNA Kit (Omega, Stamford, CT, USA).
Mitochondrial (mt) cyt b gene was used as a molecular marker for species identification. The forward primer was L14322:5′-GAC TTG AAG AAC CAC CGT TGT TAT TCA AC-3′ and the reverse primer H15576: 5′-GCG CTA GGG AGG AAT TTA ACC TCC-3′. PCR reactions had a final volume of 25 µl and contained 2.5 µl 10 ×  PCR Buffer (TaKaRa, Dalian, China), 0.2 µl of 10 mmol/L dNTPs, 1 µL of each 10 µmol/L primers, 0.5 u Taq enzyme (TaKaRa, Dalian, China), 1 µl of template DNA. The PCR reaction cycles were as follows: pre-denaturation at 94 °C for 4 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 90 s, and a final extension at 72 °C for 8 min.
PCR products were checked via 1.5% agarose gel electrophoresis and visualized with ethidium bromide to verify successful amplification. PCR DNA bands with expected size were purified using Cycle-Pure Kit (Omega, Stamford, CT, USA) and sequenced with the same primers as PCR (Tianyi biotech. Co. Ltd, Wuhan, China).

Streptavidin, 3‐Glycidoxypropyltrimethoxysilane (GPTMS), and bovine serum albumin (BSA) were obtained from Energy Chemical Co., Ltd. The UV‐cleavable oligonucleotide linkers modified with biotin and PCR primer pairs were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The water purification system was bought from ELGA (London, UK). The PCR chemicals, included 10×PCR buffer, dNTPs mixture, and Taq polymerase, were all purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. The fluorescent dye for NA was bought from Invitrogen.
In this work, silica capillaries (1.7 mm i.d. × 3.0 mm o.d.) were purchased from Guanxiu Quartz Co., Ltd. (Lianyungang, China). The semiconductor heater was fabricated by Wenext Co., Ltd. (Shenzhen, China). The temperature controller (REX C100) was obtained from Yixin Sinilink Co., Ltd. (Qingdao, China). The semiconductor heater and temperature sensor were obtained from Haoyi Thermal Electrics Factory (Guangzhou, China). The fluorescence imaging system was bought from Oeabt Optical Technology (China) Co., Ltd.