Primestar max dna polymerase

Manufactured by Takara Bio

Sourced in Japan, China, United States, France, Canada

PrimeSTAR Max DNA Polymerase is a high-fidelity DNA polymerase designed for accurate DNA amplification. It exhibits superior thermal stability and processivity, enabling efficient and accurate DNA synthesis.

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PrimeSTAR Max DNA Polymerase master mix PrimeSTAR Max DNA Polymerase reagent PrimeSTAR Max DNA polymerase system PrimeSTAR Max Premix (2×) DNA polymerase PrimeStar Max DNA polymerase premix PrimeSTAR Max DNA Polymerase kit PrimeSTAR Max polymerase PrimeSTAR DNA polymerase PrimeSTAR Max PrimeSTAR polymerase

580 protocols using primestar max dna polymerase

Targeted Circular RNA Editing

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Minigene fragments were amplified from human placenta genomic DNA (Sigma) (for intronic sequences) or EC109 cDNA (for exonic sequences) using PrimeSTAR Max DNA polymerase (Clontech) with overlapped primers and ligated into one piece of DNA, followed by ligation into pcDNA3.1+ vector. KAPA HiFi polymerase (KAPA Biosystems) was used to introduce point mutations into minigene using primers with corresponding mutation(s).
Overexpression plasmids were obtained by cloning coding sequences of protein, which were amplified by PrimeSTAR Max DNA polymerase (Clontech), into pLenti6 vector. ADARs-targeting short hairpin RNAs (shRNAs) were designed using RNAi Platform (Broad Institute) and were cloned into pLKO.1_puro plasmid using AgeI and EcoRI restriction sites.
CasRX system (pXR001: EF1a-CasRx-2A-EGFP and pXR003: CasRx gRNA cloning backbone) was a gift from Patrick Hsu (pXR001: Addgene plasmid #109049, http://n2t.net/addgene:109049, RRID:Addgene_109049; pXR003: Addgene plasmid #109053, http://n2t.net/addgene:109053, RRID:Addgene_109053)^{51 (link),52}. Guide RNA (gRNA) sequences were designed using sequence of circRNAs around BSJ with a length of 21 bp and cloned into pXR003 plasmid using BbsI restriction sites.

Shen H., An O., Ren X., Song Y., Tang S.J., Ke X.Y., Han J., Tay D.J., Ng V.H., Molias F.B., Pitcheshwar P., Leong K.W., Tan K.K., Yang H, & Chen L. (2022). ADARs act as potent regulators of circular transcriptome in cancer. Nature Communications, 13, 1508.

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Minigene Cloning and Mutagenesis Protocol

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Minigene sequences were amplified from human placenta genomic DNA (Sigma) using PrimeSTAR Max DNA polymerase (Clontech) and subcloned into pcDNA3.1 + plasmid using BamHI and XhoI restriction sites. Deletions and point mutations were introduced into minigene plasmid by PCR mutagenesis using KAPA HiFi polymerase (KAPA Biosystems) with primers containing respective mutations. Coding sequences of protein were amplified by PrimeSTAR Max DNA polymerase (Clontech) and cloned into pLenti6 vector with respective restriction sites. shRNA sequences were designed using RNAi Platform (Broad Institute) and cloned into pLKO.1_puro plasmid using AgeI and EcoRI restriction sites.

Tang S.J., Shen H., An O., Hong H., Li J., Song Y., Han J., Tay D.J., Ng V.H., Bellido Molias F., Leong K.W., Pitcheshwar P., Yang H, & Chen L. (2020). Cis- and trans-regulations of pre-mRNA splicing by RNA editing enzymes influence cancer development. Nature Communications, 11, 799.

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CRISPR-Cas9 Mutation Analysis in Tomato

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Tomato DNA was extracted using a DNA extraction kit (GeneBette, Beijing, China), and used as a template to amplify the target sites using PrimeSTAR Max DNA polymerase (TaKaRa, Ohtsu, Japan). To analyse mutation types of each T0 line, we cloned the PCR fragments into the pMD20-T vector (TaKaRa, Ohtsu, Japan), sent 15 individual clones for sequencing, and analysed the mutations. To analyse mutation types of T1 and T2 lines, the PCR products were sequenced. The primers used for amplification are shown in Table S3, see online supplementary material. To identify Cas9, the PCR products were amplified using primers specific for Cas9 (Table S4, see online supplementary material).
To analyse off-target mutations, we used CRISPR-P to predict the potential off-target sites (Table S1, see online supplementary material). The corresponding primers (Table S5, see online supplementary material) for each site were used for PCR amplification using PrimeSTAR Max DNA polymerase (TaKaRa, Ohtsu, Japan), and the PCR products were sequenced.

Huang H., Zhao W., Qiao H., Li C., Sun L., Yang R., Ma X., Ma J., Song S, & Wang S. (2022). SlWRKY45 interacts with jasmonate-ZIM domain proteins to negatively regulate defense against the root-knot nematode Meloidogyne incognita in tomato. Horticulture Research, 9, uhac197.

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Heterologous Expression of EF-P Proteins

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The E. coli Flk and GntX expression plasmids (pCA24N-derived ASKA library plasmid) were obtained from the National Bio-Resource Project (NBRP), Japan [55 (link)]. The DNA fragment encoding EarP(Nm) was PCR-amplified from N. meningitidis HT1125 genomic DNA by PrimeStar Max DNA polymerase (Takara), and cloned into pMW-NmE to construct the plasmid pMW-NmED (S1 Table). The DNA fragment encoding E. coli EF-P, EpmA, and EpmB was PCR-amplified from the plasmid pACTK-EGY [18 (link)] by PrimeStar Max DNA polymerase (Takara), and cloned into pMW119 to construct the plasmid pMW-EcEGY. The plasmids pMW119, pMW-NmE, pMW-NmED, and pMW-EcEGY were co-transformed with the Flk plasmid into E. coli BW25113 and Δefp cells. The cells harboring these plasmids were grown in LB broth (Miller) medium to an OD₆₀₀ of 0.6, and then protein expression was induced with 1 mM IPTG at 37°C for 6 hr. The cells were harvested, and the expressed Flk protein with the His₆-tag (MRGSHHHHHHTDPALRP) and the EF-P protein were analyzed by SDS-PAGE and western blotting, using an anti-His₆ antibody and a polyclonal antibody against EF-P(Ec).

Yanagisawa T., Takahashi H., Suzuki T., Masuda A., Dohmae N, & Yokoyama S. (2016). Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability. PLoS ONE, 11(2), e0147907.

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Construction and Verification of ccdC Mutant and Complement Strains in FZB42

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The ccdC sequence of FZB42 and its flanking regions were amplified with the primers szz38, szz39, and PrimeSTAR^® Max DNA Polymerase (Takara, Maebashi, Japan). The PCR product was added to a -A-tail using Taq DNA polymerase and then inserted into the commercial pMD-19 vector, resulting in the plasmid pMD-19-ccdC, which was named pSZZ11. The restriction endonuclease Nru I (Takara, Maebashi, Japan) was used to cut pSZZ11. The primers szz46 + Nru I and szz47 + Nru I were used to amplify the speR gene from pFB103. The speR gene was also cut by Nru I and then ligated to the pSZZ11 enzyme digestion product, yielding pSZZ17 (pMD-19-ccdC-speR), which was transformed into FZB42 as described previously. The transformants were verified with the primers szz01 and szz02, and the resulting strain was named SZZ15, the ccdC knockout strain.
The ccdC sequence of FZB42 and its flanking regions were amplified with the primers szz44 + Kpn I, szz45 + Cla I, and PrimeSTAR^® Max DNA Polymerase (Takara, Maebashi, Japan). The PCR product and plasmid pFB01 [65 (link)] were both cut with Cla I and then ligated to obtain pSZZ13 (pFB01-ccdC), which was transformed into FZB42 as described previously. The transformants were verified correctly with the primers FBO550 and FBO16, which were named SZZ18, the ccdC complement strain.

Shao L., Shen Z., Li M., Guan C., Fan B., Chai Y, & Zhao Y. (2024). ccdC Regulates Biofilm Dispersal in Bacillus velezensis FZB42. International Journal of Molecular Sciences, 25(10), 5201.

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Constructing Luciferase Reporters for ABCB1 Gene

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The upstream region (from bp −2091 to +701, where +1 indicates the transcription start site) and 3′ UTR (from bp +1 to +391, where +1 indicates the stop codon) of the human ABCB1 gene were amplified using PrimeSTAR MAX DNA polymerase (Takara Bio Inc). These sequences were subcloned into the pGL4.12 luciferase-reporter vector (Promega) using KpnI and NheI restriction sites and into the pGL4.13 luciferase-reporter vector (Promega) using XbaI and FseI restriction sites. The minigene sequence was amplified using PrimeSTAR MAX DNA polymerase (Takara Bio Inc). The sequence was subcloned into the pcDNA3.1 (Invitrogen; Life Technologies) vector using NheI and KpnI restriction sites. Sequences of primers are listed in Table 2.Table 2

Primer sets for plasmid construction and minigene assay

Gene	Primers
Human ABCB1 promoter
Forward for −2091 bp	5′-GCTGGTACCTCAACTTGCAAGGGGACCAG-3′
Reverse for +703 bp	5′-ATAGCTAGCCGACCTGAAGAGAAACCGCA-3′
Human ABCB1 3′ UTR
Forward for +1 bp	5′-GCGCTCTAGAAACTCTGACTGTATGAGATG-3′
Reverse for +391 bp	5′-TAAGGCCGGCCAGTCACATGAAAGTTTAG-3′
Human ABCB1 minigene
Forward for exon 27	5′-CGTGCTAGCGCAAGCTGTTAGAACTTTACTTTCA-3′
Reverse for exon 28	5′-CGCGGTACCACAGGCAGTTTGGACAAGATGA-3′

The numbers indicate the distance from the transcription site (+1) for the promoter vector or from the stop codon (+1) for the 3′ UTR vector.

Omata Y., Yamauchi T., Tsuruta A., Matsunaga N., Koyanagi S, & Ohdo S. (2021). RNA editing enzyme ADAR1 governs the circadian expression of P-glycoprotein in human renal cells by regulating alternative splicing of the ABCB1 gene. The Journal of Biological Chemistry, 296, 100601.

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Cloning and Mutagenesis of Ancestral GH19 Chitinases

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Codon-optimized genes encoding the ancestral GH19 chitinase proteins and loopful type GH19 chitinase from Secale cereale (Uniprot: Q9FRV0; residues 24-266) were synthesized by TWIST Bioscience and cloned into the pET-22b (+) vector using the iVEC3^{51 (link)}. PCR amplifications for synthetic genes and a linear-pET-22b (+) vector were performed with using PrimeSTAR® Max DNA polymerase (TaKaRa) and the designed primers (Supplementary Table 1) containing appropriate overlapping regions for iVEC3. The gene coding loopless type GH19 chitinase from Gemmabryum coronatum (Uniprot: A9ZSX9; residues 25-228) cloned into the pET-22b (+) vector was a gift from Toki Taira.
Site-directed mutagenesis was achieved by Inverse PCR using PrimeSTAR® Max DNA polymerase (TaKaRa) with the designed primers (Supplementary Table 1). Successful cloning and mutagenesis were confirmed by Sanger sequencing.

Kozome D., Sljoka A, & Laurino P. (2024). Remote loop evolution reveals a complex biological function for chitinase enzymes beyond the active site. Nature Communications, 15, 3227.

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PCR and Plasmid Purification Protocol

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PrimeSTAR Max DNA polymerase (Takara Bio), with a mismatch rate of 12 bases per 542,580 total bases, was selected to minimize errors in the PCR products. Genomic DNA fragments were purified using a MiniBEST Bacteria Genomic DNA Extraction Kit (Takara Bio) and a MiniBEST Agarose Gel DNA Extraction Kit (Takara Bio), while plasmid DNA samples were purified using a MiniBEST Plasmid Purification Kit (Takara Bio). PCR assays were performed using PrimeSTAR Max DNA polymerase (Takara Bio). Restriction and T4 DNA ligase enzymes were purchased from TransGen Biotech. The in vitro seamless assembly of different DNA fragments was performed using a pEASY-Uni Seamless Cloning and Assembly Kit (TransGen Biotech) according to the manufacturer’s instructions.

Jing W., Liu J., Wu S., Chen Q., Li X, & Liu Y. (2020). Development of a Method for Simultaneous Generation of Multiple Genetic Modification in Salmonella enterica Serovar Typhimurium. Frontiers in Genetics, 11, 563491.

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Crude DNA extraction from blastocyst

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To generate crude DNA for a PCR template, we used the approach of Sakorai et al. with some modifications.Citation 27 Briefly, 0.1 ml of KSOM medium containing 1 blastocyst was transferred with a glass micropipette to the bottom of a 0.2 ml PCR tube (Thermo Scientific, USA) under a stereomicroscope, and blastocyst lysis buffer [125 µg/ml proteinase K, 100 mM Tris-HCl (pH 8.3), 100 mM KCl, 0.02% gelatin, 0.45% Tween 20, and 60 µg/ml yeast tRNA (Ambion, USA)] was prepared. Lysis buffer (10 µl) was added to each tube. In the PCR machine (ABI GeneAmp PCR System 9700, Japan), each PCR tube was incubated at 55 °C for 60 min and then at 99 °C for 30 min. The crude DNA was kept at -20 °C until ready to use.
To obtain amplifications for blastocyst genotyping, two PCR cycles were performed. The first round of PCR was performed in a 20 µl volume comprising 10 µl of Prime STAR Max DNA Polymerase (Takara, Japan), 0.5 M forward and reverse primers, and 5 µl of crude DNA solution. After the first round of reaction, 2 µl of the sample was added to the second round of PCR mixture, which comprised 10 µl of Prime STAR Max DNA Polymerase (Takara, Japan) and 0.5 M forward and reverse primers to form a total volume of 20 µl. Using the same protocol as the previous round of PCR, amplification was carried out for 40 cycles. The sequences of the PCR primers are listed in Supplementary Table 2.

Guo X., Geng L., Jiang C., Yao W., Jin J., Liu Z, & Mu Y. (2023). Multiplexed genome engineering for porcine fetal fibroblasts with gRNA-tRNA arrays based on CRISPR/Cas9. Animal biotechnology, 34(9).

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Typing of S. aureus Protein A Gene

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The spa typing was performed by amplification of polymorphic X region of the S. aureus protein A gene (spa), using the standard primers spa-1113F (5′-TAAAGACGA-TCCTTCGGTGAGC-3′) and spa-1514R (5′-CAGCAGTAGTGCCGTTTGCTT-3′). The primers and protocol are available on the Ridom Spa Server database (http://www.spaserver.ridom.de, accessed on 3 October 2021). It was conducted according to methods described previously and modified as described herein [38 (link)]. Briefly, the PCR reaction mix contained 250 nmol of each primer, 12.5 µL of 2× PrimeSTAR^® Max DNA Polymerase (Takara Bio Inc., Dalian, China), and 1 µL of DNA template, and genomic DNA was extracted according to the manufacturer’s TIANamp Bacteria DNA Kit instructions (TianGen Biotech, Beijing, China). The PCR was performed under the following conditions: initial denaturation at 98 °C for 5 min, 32 cycles at 98 °C for 10 s, 60 °C for 15 s, and 72 °C for 30 s, and a final extension of 10 min at 72 °C. All the PCR products were sequenced by Tsingke Biotechnology Co., Ltd. (Nanjing, China), and then spa type was identified using this database.

Wang H., Shen J., Zhu C., Ma K., Fang M., Li B., Wang W, & Xue T. (2022). Antibiotics Resistance and Virulence of Staphylococcus aureus Isolates Isolated from Raw Milk from Handmade Dairy Retail Stores in Hefei City, China. Foods, 11(15), 2185.

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