Taq dna polymerase

To compare the detection efficiency, a PCR assay targeting the Salmonella invA gene was performed in parallel with LAMP primer, as shown in Table 1. In addition, SYBR green I dye was used to enhance the specificity in the qPCR reaction. The PCR amplification reaction contained 1x Taq DNA polymerase buffer, 1.2 mM dNTPs, 0.8 μM F3 and B3 primers and 8 U Taq DNA polymerase (New England Biolabs), and 5 ng of each DNA extract as a template in a final volume of 25 μL. The cycling conditions comprised of a single initial denaturation at 94°C for 2 min followed by 30 cycles at 90°C for 30 sec (denaturation), 60°C for 30 sec (annealing), 72°C for 30 sec (extension), and a final extension step at 72°C for 5 min. After the PCR amplification, the products were analyzed by electrophoresis using 1.5% agarose gel, which was stained with ethidium bromide. Then, the gels were visualized under ultraviolet light.

Paramagnetic isolation of glomeruli from both genotypes was performed as described in a previous study (40 (link)). Genomic DNA was isolated from harvested glomeruli by the addition of Direct PCR Tail Peqgold (Peqlab) and proteinase K (Qiagen), according to the manufacturer’s protocol, and incubation for 3h at 55°C and 800 rpm. Subsequent inactivation of proteinase K was achieved by heating the sample to 85°C for 45 min. End point PCR using Taq DNA Polymerase (M0273X, New England Biolabs) and primers indicated by The Jackson Laboratories for the B6.Cg-Tg(NPHS2-cre)295^Lbh/J strain verified successful excision of the floxed transgene by Nphs2-driven Cre recombinase (Supplementary Figure 1D).

All chemical reagents were of analytical grade and purchased from Sigma (St. Louis, MO). Restriction enzymes, Taq DNA polymerase, T4 ligase, and buffers for cloning were purchased from New England Biolabs, Inc. (Beverly, MA). Ultrapure agarose was purchased from GIBCO BRL-Life Technologies (Rockville, MD). Recombinant Human Glycoprotein 130 Fc Chimera (gp130) was purchased from R&D (671-GP, Bio-techne, Minneapolis, MN).
Biacore chips and the amino coupling kit were purchased from General Electric Healthcare Life Science (Piscataway, NJ). Escherichia coli DH5α and BL21 (DE3) strains were from Stratagene (La Jolla, CA, USA).

Genomic DNA from snap-frozen cell pellet samples (between passage 15 and 25) was isolated using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. To confirm the loss of episomal plasmids in the generated iPSCs, we performed a standard polymerase chain reaction (PCR) using plasmid-specific primer pairs (Table 3), which are complementary to four different regions present in all three plasmids. One PCR reaction (50 μL) was performed using 50 ng of genomic DNA, following the manufacturer’s instructions using Taq DNA Polymerase with Standard Taq Buffer (New England Biolabs, Frankfurt, Germany).
For gene expression analyses, total RNA from snap-frozen cell pellets was isolated using a NucleoSpin RNA Plus isolation kit (Macherey-Nagel, Düren, Germany), treated with RNase-free DNase (Qiagen, Hilden, Germany) and subsequently transcribed into cDNA with Omniscript RT kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The cDNA was then amplified using Taq DNA Polymerase with Standard Taq Buffer (New England Biolabs, Frankfurt, Germany). All oligonucleotides (Sigma-Aldrich) are listed in Table 3.

The gene kerBp was amplified with upstream primer (5′-CGG GAT CCA TGT GCG TTA AAA AGA AAA ATG TTA TGA CAA G-3′) and downstream primer (5′-GCA CGC GTT TAA TTT GAT GCT GCT TGC ACA TTA ATC-3′). The plasmid pMA5 was extracted and double-digested with the restriction enzyme Mlu I and Bam H. I. A randomly mutation library was constructed according to Zhang et al. (Zhang and Zhang 2011 (link)) with modifications by error-prone PCR reaction (5 mM MgCl₂, 0.2 mM MnCl₂, 0.2 mM dATP, 0.2 mM dGTP, 1 mM dCTP, 1 mM dTTP, 0.05 U/μL polymerase, and 0.4 mM each of the primers). The error-prone PCR was conducted by using the NEB Taq DNA polymerase (95 °C denaturation, 3 min; 29 cycles of 95 °C denaturation, 30 s; 57 °C annealing, 30 s; and 72 °C extension, 1.5 min, followed by 72 °C extension for 5 min). The error-prone PCR products were gel-purified and connected to the plasmid pMA5, which were tramsformed into E. coli JM109 competent cells. Plasmids obtained were finally expressed in B. subtilis WB600 and the strains with large transparent circles were selected directly. The mutant strains were cultured at 37 °C in 250-mL flasks containing 30 mL TB medium (50 µg/mL Kan^r) for 60 h and the supernatant was collected by centrifuging at 4 °C and 8000g for 20 min for keratinase activity measurement.