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TaKaRa puc18
Evaluation of the specificity of the BLV-CoCoMo-qPCR primers . (A) Real-time PCR using the CoCoMo 6 and CoCoMo 81 primers from the BLV-CoCoMo-qPCR was performed using 0.3 ng of the following infectious molecular clones: BLV (pBLV-IF, lane 2); HTLV-1 (pK30, lane 3); HIV-1 (pNL4-3, lane 4); SIV (pSIVmac239/WT, lane 5); MMTV (hybrid MMTV, lane 6); M-MLV (pL-4, lane 7); and the plasmids <t>pUC18</t> (lane 8), pUC19 (lane 9), pBR322 (lane 10), and pBluescript SK(+) (lane 11). PCR products were subjected to 3% agarose gel electrophoresis. Lane 1, DNA marker Φ × 174- Hae III digest. A PCR product 168 bp in length is indicated by an arrow. (B) The number of BLV provirus copies in 1 μg of DNA from each DNA sample is indicated by lowercase. Values represent the mean ± standard deviation (SD) of the results of three independent experiments.
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Images

1) Product Images from "BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm"

Article Title: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm

Journal: Retrovirology

doi: 10.1186/1742-4690-7-91

Evaluation of the specificity of the BLV-CoCoMo-qPCR primers . (A) Real-time PCR using the CoCoMo 6 and CoCoMo 81 primers from the BLV-CoCoMo-qPCR was performed using 0.3 ng of the following infectious molecular clones: BLV (pBLV-IF, lane 2); HTLV-1 (pK30, lane 3); HIV-1 (pNL4-3, lane 4); SIV (pSIVmac239/WT, lane 5); MMTV (hybrid MMTV, lane 6); M-MLV (pL-4, lane 7); and the plasmids pUC18 (lane 8), pUC19 (lane 9), pBR322 (lane 10), and pBluescript SK(+) (lane 11). PCR products were subjected to 3% agarose gel electrophoresis. Lane 1, DNA marker Φ × 174- Hae III digest. A PCR product 168 bp in length is indicated by an arrow. (B) The number of BLV provirus copies in 1 μg of DNA from each DNA sample is indicated by lowercase. Values represent the mean ± standard deviation (SD) of the results of three independent experiments.
Figure Legend Snippet: Evaluation of the specificity of the BLV-CoCoMo-qPCR primers . (A) Real-time PCR using the CoCoMo 6 and CoCoMo 81 primers from the BLV-CoCoMo-qPCR was performed using 0.3 ng of the following infectious molecular clones: BLV (pBLV-IF, lane 2); HTLV-1 (pK30, lane 3); HIV-1 (pNL4-3, lane 4); SIV (pSIVmac239/WT, lane 5); MMTV (hybrid MMTV, lane 6); M-MLV (pL-4, lane 7); and the plasmids pUC18 (lane 8), pUC19 (lane 9), pBR322 (lane 10), and pBluescript SK(+) (lane 11). PCR products were subjected to 3% agarose gel electrophoresis. Lane 1, DNA marker Φ × 174- Hae III digest. A PCR product 168 bp in length is indicated by an arrow. (B) The number of BLV provirus copies in 1 μg of DNA from each DNA sample is indicated by lowercase. Values represent the mean ± standard deviation (SD) of the results of three independent experiments.

Techniques Used: Real-time Polymerase Chain Reaction, Clone Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Standard Deviation

2) Product Images from "Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis"

Article Title: Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-018-1309-3

Acyl-CoA thioesterase activity of ptTES1. a ptTES1 was expressed in frame with lacZ (upper) in E. coli K27 fadD mutant fadD88 and resulted in the color change of E. coli colonies grown on M9 agar plate containing neutral red. Colonies that express an active ptTES1 are dark pink, while those containing empty vector pUC18 or expressing palmitoyl protein thioesterase ptPPT1 ( J10454 ) are light yellow. The time-dependent hydrolysis by ptTES1 of varied concentrations of decanoyl-CoA (10:0), myristoyl-CoA (14:0), and stearoyl-CoA (18:0) ( b ); palmitoyl-CoA (16:0), oleoyl-CoA (18:1), and eicosapentaenoyl-CoA (20:5) ( c ); and malonyl-CoA (3:0) ( d ); was monitored by measuring the change of A 420 . Reactions were carried out at 37 °C in mixture including 10 mM Hepes (pH 7.5), 50 mM KCl, and 0.3 mM DTNB
Figure Legend Snippet: Acyl-CoA thioesterase activity of ptTES1. a ptTES1 was expressed in frame with lacZ (upper) in E. coli K27 fadD mutant fadD88 and resulted in the color change of E. coli colonies grown on M9 agar plate containing neutral red. Colonies that express an active ptTES1 are dark pink, while those containing empty vector pUC18 or expressing palmitoyl protein thioesterase ptPPT1 ( J10454 ) are light yellow. The time-dependent hydrolysis by ptTES1 of varied concentrations of decanoyl-CoA (10:0), myristoyl-CoA (14:0), and stearoyl-CoA (18:0) ( b ); palmitoyl-CoA (16:0), oleoyl-CoA (18:1), and eicosapentaenoyl-CoA (20:5) ( c ); and malonyl-CoA (3:0) ( d ); was monitored by measuring the change of A 420 . Reactions were carried out at 37 °C in mixture including 10 mM Hepes (pH 7.5), 50 mM KCl, and 0.3 mM DTNB

Techniques Used: Activity Assay, Mutagenesis, Plasmid Preparation, Expressing

3) Product Images from "Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance"

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance

Journal: Nature Communications

doi: 10.1038/s41467-017-02304-7

Serum IL-21 and IL-33 are stimulated in B6 HDI mice but not BPS HDI mice. Sera from BPS, B6 and pUC18 HDI BALB/c mice were collected at indicated time points and levels of selected cytokines quantitated using multiplex-based assay. Group means and s.e.m. within group of serum IL-21 ( a ) and IL-33 ( b ) measurements are presented with group sizes ( n ) indicated. BPS and B6 mice data are compared against pUC18 mice and statistical significance calculated using unpaired two-tailed t -test. * p
Figure Legend Snippet: Serum IL-21 and IL-33 are stimulated in B6 HDI mice but not BPS HDI mice. Sera from BPS, B6 and pUC18 HDI BALB/c mice were collected at indicated time points and levels of selected cytokines quantitated using multiplex-based assay. Group means and s.e.m. within group of serum IL-21 ( a ) and IL-33 ( b ) measurements are presented with group sizes ( n ) indicated. BPS and B6 mice data are compared against pUC18 mice and statistical significance calculated using unpaired two-tailed t -test. * p

Techniques Used: Mouse Assay, Multiplex Assay, Two Tailed Test

Related Articles

Clone Assay:

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance
Article Snippet: Genotype D strain was a gift from Dr. Yongxiang Wang, Fudan University, and other strains were chemically synthesised. .. HDI plasmids were constructed by cloning ~1.3 fold over-length HBV genome into pUC18 (Takara, China) (Supplementary Fig. ). .. BPS/Xnull , BPS/Pnull and BPS/Cnull were created by prematurely terminating X, P and C ORFs (Supplementary Fig. ).

Article Title: Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis
Article Snippet: Escherichia coli DH5α (Transgen, China) was used for plasmid construction and propagation according to the manufacturer’s instructions. .. To functionally express ptTES1 in E. coli XL1-blue, the codon-optimized ptTES1 gene was synthesized and cloned in frame into pUC18 (Takara, China), generating the pUC18-phaeoTES1 plasmid. .. To overexpress ptTES1 in E. coli Rosetta (DE3), the codon-optimized ptTES1 gene was synthesized and cloned into pET28a (Novagen), generating the pET28a-phaeoTES1 plasmid.

Article Title: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm
Article Snippet: The Xba I-Xba I fragment including the MR1 sequence was then subcloned into pBluescript II SK (+) (Stratagene). .. Additional clones used included a BLV infectious clone, pBLV-IF [ ]; a HTLV-1 infectious clone, pK30 [ ]; a HIV-1 infectious clone, pNL4-3 [ ]; a SIV infectious clone, SIVmac239/WT [ ]; a hybrid MMTV provirus plasmid [ ]; a M-MLV infectious clone, pL-4 [ ] and plasmids including pUC18 (Takara Bio Inc., Tokyo, Japan), pUC19 (Takara Bio Inc.), and pBR322 (Promega). .. pBLV-LTR/SK and pBoLA-DRA/SK were digested with Sca I and purified using a Sephadex G-50 column (GE Healthcare Japan, Tokyo, Japan).

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: A fragment containing the full sequence of PhrdB promoter (426 bp) with a 5’ Eco RI site and a 3’ Nco I site was amplified by PCR from S . coelicolor M145 genomic DNA using primers H1/H2. .. The resulting PCR products were cloned separately into pUC18 (TaKaRa, Japan), and the products were subsequently excised from the resulting constructs using Nco I-Hin dIII and Eco RI-Nco I and then ligated with the Hin dIII-Eco RI fragment from pSPU241. .. The resulting plasmid was digested with Bgl II, and the resulting fragment containing the full sequences of kanJ , kanK and PhrdB was inserted into the Bgl II sites of pEAP1 to generate pHJK1 ( ).

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The N1 (1747 bp) and N2 (1822 bp) fragments, representing the upstream and downstream homologous arms, respectively, were amplified by polymerase chain reaction (PCR) from S . kanamyceticus CG305 genomic DNA using primers AP1III/ AP2III and AP3III/ AP4III, respectively ( ). .. N1 and N2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids using Xba I-EcoR I and Eco RI-Kpn I, respectively. .. The excised products containing the upstream and downstream fragments of kanN were then ligated with the Xba I-Kpn I fragment of pD2925 to yield pDLA302 for in-frame deletion of kanN ( ).

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The J1 (1430 bp) and J2 (1986 bp) fragments, representing the upstream and downstream homologous arms, respectively, were amplified by PCR from S . kanamyceticus CG305 genomic DNA using primers JII1/ JII2 and JII3/ JII4, respectively ( ). .. J1 and J2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids with Eco RI-Hin dIII and Eco RI-Bam HI, respectively. .. The excised products were then ligated with the Hin dIII-Bam HI fragment of pIJ2925.

Article Title: Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression
Article Snippet: Thus, to generate a complete pk1 knockout virus and to avoid any impact on pp78/83 expression, a 1,575-bp region containing part of the pp78/83 and pk1 coding regions of bMON14272 (nt 5,936 to 7,510) was replaced with a 1,038-bp chloramphenicol resistance ( CmR ) gene cassette, and then a copy of pp78/83 was inserted into the mini- att Tn 7 site via site-specific Tn7 transposition ( ). .. The CmR gene cassette was PCR amplified from pUC18-Cm ( ) with primer pair CmRU/CmRD (all PCR primers are listed in ) and cloned into pUC18 (TaKaRa) to generate pUC18-CmR. .. Two homologous fragments flanking the deletion region were PCR amplified from bMON14272 using primer pairs pp78/83-U/pp78/83-D and pk1-U/pk1-D.

Article Title: Pathway engineering of Propionibacterium jensenii for improved production of propionic acid
Article Snippet: P. jensenii ATCC 4868 and K. pneumoniae subsp. pneumoniae ATCC 12657 were purchased from the American Type Culture Collection (ATCC) and used as templates to amplify genes or as hosts for gene expression and deletion. .. E. coli JM110 (Stratagene, La Jolla, CA) was used for plasmid cloning and maintenance. pUC18 (TaKaRa, Dalian, China) was used as suicide plasmid to knock out genes in P. jensenii , and the shuttle vector pZGX04 was constructed in our previous study . pRS303 was maintained in our laboratory to amplify the hygromycin B resistance gene (hygB ). .. P. jensenii was cultured anaerobically at 32 °C in sodium lactate broth medium (10 g·L−1 yeast extract, 10 g·L−1 tryptic soy broth, and 10 g·L−1 sodium lactate), and E. coli and K. pneumoniae were grown at 37 °C in Luria-Bertani medium (10 g·L−1 NaCl, 10 g·L−1 peptone, and 5 g·L−1 yeast extract) with a shaking speed of 220 rpm.

Article Title: A Rice gid1 Suppressor Mutant Reveals That Gibberellin Is Not Always Required for Interaction between Its Receptor, GID1, and DELLA Proteins
Article Snippet: A genomic GID1 fragment including GID1 exon 1 was amplified from gDNA of soybean using primer sets [ -(8) online, colored in yellow], and GID1 exon 2 was amplified from genomic DNA of soybean using primer sets [see -(8) online, colored in purple]. .. The amplified first and second exons of each soybean gene were joined using restriction enzyme sites and cloned into pUC18 (TAKARA) at restriction enzyme sites shown in -(8) online. .. Inverted PCR was then performed using primer sets [see -(9) online].

Article Title: Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli
Article Snippet: The full-length sequence of E. coli tesA was amplified and cloned into plasmid pUC18S. .. Plasmid pUC18S was created by replacing the Aat II/Eam 1105I fragment (containing the ampicillin-resistance gene) of pUC18 (Takara) with an Aat II/Eam 1105I fragment (containing the spectinomycin-resistance gene) amplified, by PCR, from pAPNC213 [ ].

Article Title: Improved Adsorption of an Enterococcus faecalis Bacteriophage ?EF24C with a Spontaneous Point Mutation
Article Snippet: Escherichia coli strains DH5α and BL21 were used for cloning and protein overexpression. .. E. faecalis phage ΦEF24C is described elsewhere – . pUC18 and pCold II plasmids were purchased from Takara Bio (Kyoto, Japan) for the purposes of cloning and protein overexpression, respectively. .. Tryptic soy broth (TSB) was used as a culture medium for E. faecalis and phage ΦEF24C.

Article Title: A gonococcal homologue of meningococcal ?-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent
Article Snippet: Phylogenetic analyses were performed by constructing a distance matrix of nucleotide mismatches using the web site of the Belozersky Institute at Moscow State University [ ] and visualized by Split decomposition analysis with the program SPLITSTREE, version 3.2 [ ]. .. HT1195 (NIID103 Δggh::spc ) and HT1196 (NIID106 Δggh::spc ), in which a spectinomycin resistance gene (spc ) was inserted into the ggh gene, were constructed as follows: A 2-kb fragment containing the ggh gene of NIID103 or NIID106 was amplified by PCR and cloned in the Sma I site of pUC18 (Takara Bio) to construct pHT412 or pHT413, respectively. .. A blunted 1-kb fragment containing the spc gene [ ] was inserted into the Eco RV sites of pHT412 and pHT413, respectively.

Article Title: Fifty-Year Trend Towards Suppression of Wolbachia-Induced Male-Killing by Its Butterfly Host, Hypolimnas bolina
Article Snippet: The PCR products were purified using a MinElute Gel Extraction Kit (QIAGEN), Alkaline Phosphatase Exonuclease I (shrimp) (Takara), a Suprec-02 spin-filter (Takara), or a Takara DNA Fragment Purification Kit (Takara). .. For cloning, some PCR products were cloned into pUC118 (Takara) or pUC18 (Takara) vectors and then used to transform Escherichia coli JM 109 or DH5α competent cells. .. The plasmids in the cells were purified using a Miniprep DNA Purification Kit (Takara), or a TaKaRa MiniBEST Plasmid Purification Kit ver.2.0 (Takara).

Amplification:

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: A fragment containing the full sequence of PhrdB promoter (426 bp) with a 5’ Eco RI site and a 3’ Nco I site was amplified by PCR from S . coelicolor M145 genomic DNA using primers H1/H2. .. The resulting PCR products were cloned separately into pUC18 (TaKaRa, Japan), and the products were subsequently excised from the resulting constructs using Nco I-Hin dIII and Eco RI-Nco I and then ligated with the Hin dIII-Eco RI fragment from pSPU241.

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The N1 (1747 bp) and N2 (1822 bp) fragments, representing the upstream and downstream homologous arms, respectively, were amplified by polymerase chain reaction (PCR) from S . kanamyceticus CG305 genomic DNA using primers AP1III/ AP2III and AP3III/ AP4III, respectively ( ). .. N1 and N2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids using Xba I-EcoR I and Eco RI-Kpn I, respectively.

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The J1 (1430 bp) and J2 (1986 bp) fragments, representing the upstream and downstream homologous arms, respectively, were amplified by PCR from S . kanamyceticus CG305 genomic DNA using primers JII1/ JII2 and JII3/ JII4, respectively ( ). .. J1 and J2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids with Eco RI-Hin dIII and Eco RI-Bam HI, respectively.

Article Title: Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression
Article Snippet: Thus, to generate a complete pk1 knockout virus and to avoid any impact on pp78/83 expression, a 1,575-bp region containing part of the pp78/83 and pk1 coding regions of bMON14272 (nt 5,936 to 7,510) was replaced with a 1,038-bp chloramphenicol resistance ( CmR ) gene cassette, and then a copy of pp78/83 was inserted into the mini- att Tn 7 site via site-specific Tn7 transposition ( ). .. The CmR gene cassette was PCR amplified from pUC18-Cm ( ) with primer pair CmRU/CmRD (all PCR primers are listed in ) and cloned into pUC18 (TaKaRa) to generate pUC18-CmR. .. Two homologous fragments flanking the deletion region were PCR amplified from bMON14272 using primer pairs pp78/83-U/pp78/83-D and pk1-U/pk1-D.

Article Title: Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies
Article Snippet: The gp37 (CpGV orf13 ) ( ) and en4 (nucleotides [nt] 616 to 1499 of AgseGV enhancin [GenBank accession no. ]) sequences were PCR amplified from the genomic DNA of the corresponding virus by using the gp37-F/gp37-R or en4-F/en4-R primer pair ( ). .. The amplified 5′ and 3′ polyhedrin segments were analyzed by agarose gel electrophoresis and then recovered from the gel by use of a gel extraction kit (Omega Bio-Tek, Norcross, GA, USA), and they were digested with NheI/SphI and EcoRI/XbaI, respectively, and then ligated into pMD18T and pUC18 (TaKaRa), respectively. .. The resulting constructs are here called pMD18T-phN110, pMD18T-phN150, pMD18T-phN170, and pMD18T-phN204 (pMD18T constructs) and pUC18-phC135, pUC18-phC95, pUC18-phC75, and pUC18-phC41 (pUC18 constructs).

Article Title: A Rice gid1 Suppressor Mutant Reveals That Gibberellin Is Not Always Required for Interaction between Its Receptor, GID1, and DELLA Proteins
Article Snippet: A genomic GID1 fragment including GID1 exon 1 was amplified from gDNA of soybean using primer sets [ -(8) online, colored in yellow], and GID1 exon 2 was amplified from genomic DNA of soybean using primer sets [see -(8) online, colored in purple]. .. The amplified first and second exons of each soybean gene were joined using restriction enzyme sites and cloned into pUC18 (TAKARA) at restriction enzyme sites shown in -(8) online. .. Inverted PCR was then performed using primer sets [see -(9) online].

Article Title: Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli
Article Snippet: The full-length sequence of E. coli tesA was amplified and cloned into plasmid pUC18S. .. Plasmid pUC18S was created by replacing the Aat II/Eam 1105I fragment (containing the ampicillin-resistance gene) of pUC18 (Takara) with an Aat II/Eam 1105I fragment (containing the spectinomycin-resistance gene) amplified, by PCR, from pAPNC213 [ ]. .. B. subtilis strains were grown in Luria-Bertani (LB) medium, or DSM, supplemented with chloramphenicol (5 μg/ml), kanamycin (5 μg/ml), or erythromycin (0.5 μg/ml), as appropriate.E. coli strains were grown in LB or M9 minimal media, supplemented with ampicillin (50 μg/ml), chloramphenicol (5 μg/ml), spectinomycin (50 μg/ml), or kanamycin (25 μg/ml), as appropriate.

Article Title: A gonococcal homologue of meningococcal ?-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent
Article Snippet: Phylogenetic analyses were performed by constructing a distance matrix of nucleotide mismatches using the web site of the Belozersky Institute at Moscow State University [ ] and visualized by Split decomposition analysis with the program SPLITSTREE, version 3.2 [ ]. .. HT1195 (NIID103 Δggh::spc ) and HT1196 (NIID106 Δggh::spc ), in which a spectinomycin resistance gene (spc ) was inserted into the ggh gene, were constructed as follows: A 2-kb fragment containing the ggh gene of NIID103 or NIID106 was amplified by PCR and cloned in the Sma I site of pUC18 (Takara Bio) to construct pHT412 or pHT413, respectively. .. A blunted 1-kb fragment containing the spc gene [ ] was inserted into the Eco RV sites of pHT412 and pHT413, respectively.

Article Title: Fifty-Year Trend Towards Suppression of Wolbachia-Induced Male-Killing by Its Butterfly Host, Hypolimnas bolina
Article Snippet: In addition, wsp ( ) was amplified from each of 3 DNA samples from H. bolina ID numbers 5, 8, and 11 and sequenced by PCR clone sequencing, and in the sample from H. bolina ID number 8, a wider region of ftsZ ( ) than that used in the MLST was also sequenced directly. .. For cloning, some PCR products were cloned into pUC118 (Takara) or pUC18 (Takara) vectors and then used to transform Escherichia coli JM 109 or DH5α competent cells.

Synthesized:

Article Title: Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis
Article Snippet: Escherichia coli DH5α (Transgen, China) was used for plasmid construction and propagation according to the manufacturer’s instructions. .. To functionally express ptTES1 in E. coli XL1-blue, the codon-optimized ptTES1 gene was synthesized and cloned in frame into pUC18 (Takara, China), generating the pUC18-phaeoTES1 plasmid. .. To overexpress ptTES1 in E. coli Rosetta (DE3), the codon-optimized ptTES1 gene was synthesized and cloned into pET28a (Novagen), generating the pET28a-phaeoTES1 plasmid.

Article Title: A Rice gid1 Suppressor Mutant Reveals That Gibberellin Is Not Always Required for Interaction between Its Receptor, GID1, and DELLA Proteins
Article Snippet: GID1 cDNAs from various species for Y2H assays were produced by PCR or synthesized based on the database sequences of mRNA or predicted coding sequences. cDNAs of grape ( Vitis vinifera ) GID1a , grape GID1b , and Brassica napus GID1 were synthesized with Eco RI- Bam HI, Eco RI- Bam HI and Eco RI- Sma I sites, respectively (GenScript), and cloned into pGBKT7. cDNAs of soybean GID1a-1 , a-2 , b-1 , b-2 , and b-3 were produced as follows. .. The amplified first and second exons of each soybean gene were joined using restriction enzyme sites and cloned into pUC18 (TAKARA) at restriction enzyme sites shown in -(8) online.

Construct:

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance
Article Snippet: Genotype D strain was a gift from Dr. Yongxiang Wang, Fudan University, and other strains were chemically synthesised. .. HDI plasmids were constructed by cloning ~1.3 fold over-length HBV genome into pUC18 (Takara, China) (Supplementary Fig. ). .. BPS/Xnull , BPS/Pnull and BPS/Cnull were created by prematurely terminating X, P and C ORFs (Supplementary Fig. ).

Article Title: Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis
Article Snippet: To functionally express ptTES1 in E. coli XL1-blue, the codon-optimized ptTES1 gene was synthesized and cloned in frame into pUC18 (Takara, China), generating the pUC18-phaeoTES1 plasmid. .. To overexpress ptTES1 in E. coli Rosetta (DE3), the codon-optimized ptTES1 gene was synthesized and cloned into pET28a (Novagen), generating the pET28a-phaeoTES1 plasmid.

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: A fragment containing the full sequence of PhrdB promoter (426 bp) with a 5’ Eco RI site and a 3’ Nco I site was amplified by PCR from S . coelicolor M145 genomic DNA using primers H1/H2. .. The resulting PCR products were cloned separately into pUC18 (TaKaRa, Japan), and the products were subsequently excised from the resulting constructs using Nco I-Hin dIII and Eco RI-Nco I and then ligated with the Hin dIII-Eco RI fragment from pSPU241. .. The resulting plasmid was digested with Bgl II, and the resulting fragment containing the full sequences of kanJ , kanK and PhrdB was inserted into the Bgl II sites of pEAP1 to generate pHJK1 ( ).

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The plasmid pDLA302 containing kanN with an in-frame deletion was constructed as follows. .. N1 and N2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids using Xba I-EcoR I and Eco RI-Kpn I, respectively.

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The plasmid pLDJ202 containing kanJ with in-frame deletion was constructed as follows. .. J1 and J2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids with Eco RI-Hin dIII and Eco RI-Bam HI, respectively.

Article Title: Pathway engineering of Propionibacterium jensenii for improved production of propionic acid
Article Snippet: P. jensenii ATCC 4868 and K. pneumoniae subsp. pneumoniae ATCC 12657 were purchased from the American Type Culture Collection (ATCC) and used as templates to amplify genes or as hosts for gene expression and deletion. .. E. coli JM110 (Stratagene, La Jolla, CA) was used for plasmid cloning and maintenance. pUC18 (TaKaRa, Dalian, China) was used as suicide plasmid to knock out genes in P. jensenii , and the shuttle vector pZGX04 was constructed in our previous study . pRS303 was maintained in our laboratory to amplify the hygromycin B resistance gene (hygB ). .. P. jensenii was cultured anaerobically at 32 °C in sodium lactate broth medium (10 g·L−1 yeast extract, 10 g·L−1 tryptic soy broth, and 10 g·L−1 sodium lactate), and E. coli and K. pneumoniae were grown at 37 °C in Luria-Bertani medium (10 g·L−1 NaCl, 10 g·L−1 peptone, and 5 g·L−1 yeast extract) with a shaking speed of 220 rpm.

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: NIH3T3 PKR-deficient cells ( ) and NIH3T3 wild-type cells were grown as described for HeLa cells. .. For experiments using EBV gene-derived constructs, transfections were performed in 100 mm plates using calcium phosphate with 15 µg of total DNA (0.5 µg of pTRE2-Flag.BDLF1, 0.25 µg of pCI-Flag.EB2 and 0.5 µg of pTet-On or pTet-Off expression vectors (Clontech) and pUC18 up to 15 µg). .. When necessary, doxycyclin was added at a concentration of 1 μg/ml.

Article Title: Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies
Article Snippet: The amplified 5′ and 3′ polyhedrin segments were analyzed by agarose gel electrophoresis and then recovered from the gel by use of a gel extraction kit (Omega Bio-Tek, Norcross, GA, USA), and they were digested with NheI/SphI and EcoRI/XbaI, respectively, and then ligated into pMD18T and pUC18 (TaKaRa), respectively. .. The resulting constructs are here called pMD18T-phN110, pMD18T-phN150, pMD18T-phN170, and pMD18T-phN204 (pMD18T constructs) and pUC18-phC135, pUC18-phC95, pUC18-phC75, and pUC18-phC41 (pUC18 constructs).

Article Title: Effects of different interchain linkers on biological activity of an anti-prostate cancer single-chain bispecific antibody
Article Snippet: Composition of the interchain linker may affect the function of the scBsAb; therefore, rational selection and design of the linker are crucial for construction of an ideal engineered antibody. .. The pUC18 and pSectag2B plasmids, restriction endonucleases, and DNA ligase were all purchased from TaKaRa Bio Inc. (Tokyo, Japan); plasmids pUC18-γ-Sm ScFv and pUC18-CD3 ScFv were constructed and stored in our laboratory. .. JM109 competent cells and HeLa cells were purchased from Tiangen Biotech Co., Ltd. (Beijing, China).

Article Title: A gonococcal homologue of meningococcal ?-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent
Article Snippet: Phylogenetic analyses were performed by constructing a distance matrix of nucleotide mismatches using the web site of the Belozersky Institute at Moscow State University [ ] and visualized by Split decomposition analysis with the program SPLITSTREE, version 3.2 [ ]. .. HT1195 (NIID103 Δggh::spc ) and HT1196 (NIID106 Δggh::spc ), in which a spectinomycin resistance gene (spc ) was inserted into the ggh gene, were constructed as follows: A 2-kb fragment containing the ggh gene of NIID103 or NIID106 was amplified by PCR and cloned in the Sma I site of pUC18 (Takara Bio) to construct pHT412 or pHT413, respectively. .. A blunted 1-kb fragment containing the spc gene [ ] was inserted into the Eco RV sites of pHT412 and pHT413, respectively.

Enzyme-linked Immunosorbent Assay:

Article Title: Effects of different interchain linkers on biological activity of an anti-prostate cancer single-chain bispecific antibody
Article Snippet: The pUC18 and pSectag2B plasmids, restriction endonucleases, and DNA ligase were all purchased from TaKaRa Bio Inc. (Tokyo, Japan); plasmids pUC18-γ-Sm ScFv and pUC18-CD3 ScFv were constructed and stored in our laboratory. .. The Lipofectamine2000 transfection kit was from Invitrogen Corporation (CA, USA).

Incubation:

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: N1 and N2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids using Xba I-EcoR I and Eco RI-Kpn I, respectively. .. First, the disruption plasmid pDLA302 was introduced into E . coli ET12567/pUZ8002 via the CaCl2 method and then into S . kanamyceticus CG305 by conjugational transfer [ ].

Article Title: Molecular insights into replication initiation by Qβ replicase using ribosomal protein S1
Article Snippet: The plasmid (pT7QB), bearing the T7 promoter and the DNA corresponding to the Qβ positive strand RNA in pUC18, was purchased from Takara, Japan. .. The plasmid (pT7QB), bearing the T7 promoter and the DNA corresponding to the Qβ positive strand RNA in pUC18, was purchased from Takara, Japan.

Luciferase:

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: For experiments using EBV gene-derived constructs, transfections were performed in 100 mm plates using calcium phosphate with 15 µg of total DNA (0.5 µg of pTRE2-Flag.BDLF1, 0.25 µg of pCI-Flag.EB2 and 0.5 µg of pTet-On or pTet-Off expression vectors (Clontech) and pUC18 up to 15 µg). .. When necessary, doxycyclin was added at a concentration of 1 μg/ml.

Expressing:

Article Title: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm
Article Snippet: To generate pBoLA-DRA/SK, which includes a full-length bovine DRA gene, we digested MR1 from the mammalian expression vector pCDM8 [ ] with Xba I. .. Additional clones used included a BLV infectious clone, pBLV-IF [ ]; a HTLV-1 infectious clone, pK30 [ ]; a HIV-1 infectious clone, pNL4-3 [ ]; a SIV infectious clone, SIVmac239/WT [ ]; a hybrid MMTV provirus plasmid [ ]; a M-MLV infectious clone, pL-4 [ ] and plasmids including pUC18 (Takara Bio Inc., Tokyo, Japan), pUC19 (Takara Bio Inc.), and pBR322 (Promega).

Article Title: Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression
Article Snippet: Thus, to generate a complete pk1 knockout virus and to avoid any impact on pp78/83 expression, a 1,575-bp region containing part of the pp78/83 and pk1 coding regions of bMON14272 (nt 5,936 to 7,510) was replaced with a 1,038-bp chloramphenicol resistance ( CmR ) gene cassette, and then a copy of pp78/83 was inserted into the mini- att Tn 7 site via site-specific Tn7 transposition ( ). .. The CmR gene cassette was PCR amplified from pUC18-Cm ( ) with primer pair CmRU/CmRD (all PCR primers are listed in ) and cloned into pUC18 (TaKaRa) to generate pUC18-CmR.

Article Title: Pathway engineering of Propionibacterium jensenii for improved production of propionic acid
Article Snippet: P. jensenii ATCC 4868 and K. pneumoniae subsp. pneumoniae ATCC 12657 were purchased from the American Type Culture Collection (ATCC) and used as templates to amplify genes or as hosts for gene expression and deletion. .. E. coli JM110 (Stratagene, La Jolla, CA) was used for plasmid cloning and maintenance. pUC18 (TaKaRa, Dalian, China) was used as suicide plasmid to knock out genes in P. jensenii , and the shuttle vector pZGX04 was constructed in our previous study . pRS303 was maintained in our laboratory to amplify the hygromycin B resistance gene (hygB ).

Article Title: Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1
Article Snippet: The used strains and vectors were of E. coli DH5α, E. coli BL21 (DE3) (Tiangen), pUC18 (Takara), and pET30a(+) (Novagen). .. Mineral salt medium that containing 1.0 g·L−1 of NH4 NO3 , 0.5 g·L−1 of NaCl, 0.5 g·L−1 of (NH4 )2 SO4 , 0.5 g·L−1 of KH2 PO4 , and 1.5 g·L−1 of K2 HPO4 was used for library screening.

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: NIH3T3 PKR-deficient cells ( ) and NIH3T3 wild-type cells were grown as described for HeLa cells. .. For experiments using EBV gene-derived constructs, transfections were performed in 100 mm plates using calcium phosphate with 15 µg of total DNA (0.5 µg of pTRE2-Flag.BDLF1, 0.25 µg of pCI-Flag.EB2 and 0.5 µg of pTet-On or pTet-Off expression vectors (Clontech) and pUC18 up to 15 µg). .. When necessary, doxycyclin was added at a concentration of 1 μg/ml.

Modification:

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: HeLa and HEK293T cells were grown in Dulbecco's modified Eagle medium supplemented with penicillin, streptomycin and 5% fetal calf serum (Invitrogen). .. For experiments using EBV gene-derived constructs, transfections were performed in 100 mm plates using calcium phosphate with 15 µg of total DNA (0.5 µg of pTRE2-Flag.BDLF1, 0.25 µg of pCI-Flag.EB2 and 0.5 µg of pTet-On or pTet-Off expression vectors (Clontech) and pUC18 up to 15 µg).

Transformation Assay:

Article Title: A gonococcal homologue of meningococcal ?-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent
Article Snippet: HT1195 (NIID103 Δggh::spc ) and HT1196 (NIID106 Δggh::spc ), in which a spectinomycin resistance gene (spc ) was inserted into the ggh gene, were constructed as follows: A 2-kb fragment containing the ggh gene of NIID103 or NIID106 was amplified by PCR and cloned in the Sma I site of pUC18 (Takara Bio) to construct pHT412 or pHT413, respectively. .. HT1195 (NIID103 Δggh::spc ) and HT1196 (NIID106 Δggh::spc ), in which a spectinomycin resistance gene (spc ) was inserted into the ggh gene, were constructed as follows: A 2-kb fragment containing the ggh gene of NIID103 or NIID106 was amplified by PCR and cloned in the Sma I site of pUC18 (Takara Bio) to construct pHT412 or pHT413, respectively.

Over Expression:

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The pHJK1 plasmid containing the PhrdB promoter, kanJ and kanK was constructed for overexpression. .. The resulting PCR products were cloned separately into pUC18 (TaKaRa, Japan), and the products were subsequently excised from the resulting constructs using Nco I-Hin dIII and Eco RI-Nco I and then ligated with the Hin dIII-Eco RI fragment from pSPU241.

Article Title: Improved Adsorption of an Enterococcus faecalis Bacteriophage ?EF24C with a Spontaneous Point Mutation
Article Snippet: Escherichia coli strains DH5α and BL21 were used for cloning and protein overexpression. .. E. faecalis phage ΦEF24C is described elsewhere – . pUC18 and pCold II plasmids were purchased from Takara Bio (Kyoto, Japan) for the purposes of cloning and protein overexpression, respectively. .. Tryptic soy broth (TSB) was used as a culture medium for E. faecalis and phage ΦEF24C.

Transfection:

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: NIH3T3 PKR-deficient cells ( ) and NIH3T3 wild-type cells were grown as described for HeLa cells. .. For experiments using EBV gene-derived constructs, transfections were performed in 100 mm plates using calcium phosphate with 15 µg of total DNA (0.5 µg of pTRE2-Flag.BDLF1, 0.25 µg of pCI-Flag.EB2 and 0.5 µg of pTet-On or pTet-Off expression vectors (Clontech) and pUC18 up to 15 µg). .. When necessary, doxycyclin was added at a concentration of 1 μg/ml.

Article Title: Effects of different interchain linkers on biological activity of an anti-prostate cancer single-chain bispecific antibody
Article Snippet: The pUC18 and pSectag2B plasmids, restriction endonucleases, and DNA ligase were all purchased from TaKaRa Bio Inc. (Tokyo, Japan); plasmids pUC18-γ-Sm ScFv and pUC18-CD3 ScFv were constructed and stored in our laboratory. .. The pUC18 and pSectag2B plasmids, restriction endonucleases, and DNA ligase were all purchased from TaKaRa Bio Inc. (Tokyo, Japan); plasmids pUC18-γ-Sm ScFv and pUC18-CD3 ScFv were constructed and stored in our laboratory.

Library Screening:

Article Title: Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1
Article Snippet: The used strains and vectors were of E. coli DH5α, E. coli BL21 (DE3) (Tiangen), pUC18 (Takara), and pET30a(+) (Novagen). .. The genomic library of Ochrobactrum anthropi YZ-1 was previously constructed and preserved in our laboratory .

Cell Culture:

Article Title: Improved Adsorption of an Enterococcus faecalis Bacteriophage ?EF24C with a Spontaneous Point Mutation
Article Snippet: E. faecalis phage ΦEF24C is described elsewhere – . pUC18 and pCold II plasmids were purchased from Takara Bio (Kyoto, Japan) for the purposes of cloning and protein overexpression, respectively. .. E. faecalis phage ΦEF24C is described elsewhere – . pUC18 and pCold II plasmids were purchased from Takara Bio (Kyoto, Japan) for the purposes of cloning and protein overexpression, respectively.

Generated:

Article Title: Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies
Article Snippet: The amplified 5′ and 3′ polyhedrin segments were analyzed by agarose gel electrophoresis and then recovered from the gel by use of a gel extraction kit (Omega Bio-Tek, Norcross, GA, USA), and they were digested with NheI/SphI and EcoRI/XbaI, respectively, and then ligated into pMD18T and pUC18 (TaKaRa), respectively. .. The amplified 5′ and 3′ polyhedrin segments were analyzed by agarose gel electrophoresis and then recovered from the gel by use of a gel extraction kit (Omega Bio-Tek, Norcross, GA, USA), and they were digested with NheI/SphI and EcoRI/XbaI, respectively, and then ligated into pMD18T and pUC18 (TaKaRa), respectively.

DNA Sequencing:

Article Title: Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis
Article Snippet: To functionally express ptTES1 in E. coli XL1-blue, the codon-optimized ptTES1 gene was synthesized and cloned in frame into pUC18 (Takara, China), generating the pUC18-phaeoTES1 plasmid. .. To overexpress ptTES1 in E. coli Rosetta (DE3), the codon-optimized ptTES1 gene was synthesized and cloned into pET28a (Novagen), generating the pET28a-phaeoTES1 plasmid.

Polymerase Chain Reaction:

Article Title: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm
Article Snippet: PCR products were cloned into pBluescript II SK (+) (Stratagene, La Jolla, CA). .. Additional clones used included a BLV infectious clone, pBLV-IF [ ]; a HTLV-1 infectious clone, pK30 [ ]; a HIV-1 infectious clone, pNL4-3 [ ]; a SIV infectious clone, SIVmac239/WT [ ]; a hybrid MMTV provirus plasmid [ ]; a M-MLV infectious clone, pL-4 [ ] and plasmids including pUC18 (Takara Bio Inc., Tokyo, Japan), pUC19 (Takara Bio Inc.), and pBR322 (Promega).

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: A fragment containing the full sequence of PhrdB promoter (426 bp) with a 5’ Eco RI site and a 3’ Nco I site was amplified by PCR from S . coelicolor M145 genomic DNA using primers H1/H2. .. The resulting PCR products were cloned separately into pUC18 (TaKaRa, Japan), and the products were subsequently excised from the resulting constructs using Nco I-Hin dIII and Eco RI-Nco I and then ligated with the Hin dIII-Eco RI fragment from pSPU241. .. The resulting plasmid was digested with Bgl II, and the resulting fragment containing the full sequences of kanJ , kanK and PhrdB was inserted into the Bgl II sites of pEAP1 to generate pHJK1 ( ).

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The N1 (1747 bp) and N2 (1822 bp) fragments, representing the upstream and downstream homologous arms, respectively, were amplified by polymerase chain reaction (PCR) from S . kanamyceticus CG305 genomic DNA using primers AP1III/ AP2III and AP3III/ AP4III, respectively ( ). .. N1 and N2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids using Xba I-EcoR I and Eco RI-Kpn I, respectively.

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The J1 (1430 bp) and J2 (1986 bp) fragments, representing the upstream and downstream homologous arms, respectively, were amplified by PCR from S . kanamyceticus CG305 genomic DNA using primers JII1/ JII2 and JII3/ JII4, respectively ( ). .. J1 and J2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids with Eco RI-Hin dIII and Eco RI-Bam HI, respectively.

Article Title: Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression
Article Snippet: Thus, to generate a complete pk1 knockout virus and to avoid any impact on pp78/83 expression, a 1,575-bp region containing part of the pp78/83 and pk1 coding regions of bMON14272 (nt 5,936 to 7,510) was replaced with a 1,038-bp chloramphenicol resistance ( CmR ) gene cassette, and then a copy of pp78/83 was inserted into the mini- att Tn 7 site via site-specific Tn7 transposition ( ). .. The CmR gene cassette was PCR amplified from pUC18-Cm ( ) with primer pair CmRU/CmRD (all PCR primers are listed in ) and cloned into pUC18 (TaKaRa) to generate pUC18-CmR. .. Two homologous fragments flanking the deletion region were PCR amplified from bMON14272 using primer pairs pp78/83-U/pp78/83-D and pk1-U/pk1-D.

Article Title: A Rice gid1 Suppressor Mutant Reveals That Gibberellin Is Not Always Required for Interaction between Its Receptor, GID1, and DELLA Proteins
Article Snippet: GID1 cDNAs from various species for Y2H assays were produced by PCR or synthesized based on the database sequences of mRNA or predicted coding sequences. cDNAs of grape ( Vitis vinifera ) GID1a , grape GID1b , and Brassica napus GID1 were synthesized with Eco RI- Bam HI, Eco RI- Bam HI and Eco RI- Sma I sites, respectively (GenScript), and cloned into pGBKT7. cDNAs of soybean GID1a-1 , a-2 , b-1 , b-2 , and b-3 were produced as follows. .. The amplified first and second exons of each soybean gene were joined using restriction enzyme sites and cloned into pUC18 (TAKARA) at restriction enzyme sites shown in -(8) online.

Article Title: Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli
Article Snippet: The full-length sequence of E. coli tesA was amplified and cloned into plasmid pUC18S. .. Plasmid pUC18S was created by replacing the Aat II/Eam 1105I fragment (containing the ampicillin-resistance gene) of pUC18 (Takara) with an Aat II/Eam 1105I fragment (containing the spectinomycin-resistance gene) amplified, by PCR, from pAPNC213 [ ]. .. B. subtilis strains were grown in Luria-Bertani (LB) medium, or DSM, supplemented with chloramphenicol (5 μg/ml), kanamycin (5 μg/ml), or erythromycin (0.5 μg/ml), as appropriate.E. coli strains were grown in LB or M9 minimal media, supplemented with ampicillin (50 μg/ml), chloramphenicol (5 μg/ml), spectinomycin (50 μg/ml), or kanamycin (25 μg/ml), as appropriate.

Article Title: A gonococcal homologue of meningococcal ?-glutamyl transpeptidase gene is a new type of bacterial pseudogene that is transcriptionally active but phenotypically silent
Article Snippet: Phylogenetic analyses were performed by constructing a distance matrix of nucleotide mismatches using the web site of the Belozersky Institute at Moscow State University [ ] and visualized by Split decomposition analysis with the program SPLITSTREE, version 3.2 [ ]. .. HT1195 (NIID103 Δggh::spc ) and HT1196 (NIID106 Δggh::spc ), in which a spectinomycin resistance gene (spc ) was inserted into the ggh gene, were constructed as follows: A 2-kb fragment containing the ggh gene of NIID103 or NIID106 was amplified by PCR and cloned in the Sma I site of pUC18 (Takara Bio) to construct pHT412 or pHT413, respectively. .. A blunted 1-kb fragment containing the spc gene [ ] was inserted into the Eco RV sites of pHT412 and pHT413, respectively.

Article Title: Fifty-Year Trend Towards Suppression of Wolbachia-Induced Male-Killing by Its Butterfly Host, Hypolimnas bolina
Article Snippet: The PCR products were purified using a MinElute Gel Extraction Kit (QIAGEN), Alkaline Phosphatase Exonuclease I (shrimp) (Takara), a Suprec-02 spin-filter (Takara), or a Takara DNA Fragment Purification Kit (Takara). .. For cloning, some PCR products were cloned into pUC118 (Takara) or pUC18 (Takara) vectors and then used to transform Escherichia coli JM 109 or DH5α competent cells. .. The plasmids in the cells were purified using a Miniprep DNA Purification Kit (Takara), or a TaKaRa MiniBEST Plasmid Purification Kit ver.2.0 (Takara).

Recombinant:

Article Title: Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1
Article Snippet: The used strains and vectors were of E. coli DH5α, E. coli BL21 (DE3) (Tiangen), pUC18 (Takara), and pET30a(+) (Novagen). .. Mineral salt medium that containing 1.0 g·L−1 of NH4 NO3 , 0.5 g·L−1 of NaCl, 0.5 g·L−1 of (NH4 )2 SO4 , 0.5 g·L−1 of KH2 PO4 , and 1.5 g·L−1 of K2 HPO4 was used for library screening.

Article Title: Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies
Article Snippet: The amplified 5′ and 3′ polyhedrin segments were analyzed by agarose gel electrophoresis and then recovered from the gel by use of a gel extraction kit (Omega Bio-Tek, Norcross, GA, USA), and they were digested with NheI/SphI and EcoRI/XbaI, respectively, and then ligated into pMD18T and pUC18 (TaKaRa), respectively. .. The resulting constructs are here called pMD18T-phN110, pMD18T-phN150, pMD18T-phN170, and pMD18T-phN204 (pMD18T constructs) and pUC18-phC135, pUC18-phC95, pUC18-phC75, and pUC18-phC41 (pUC18 constructs).

Mutagenesis:

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance
Article Snippet: HDI plasmids were constructed by cloning ~1.3 fold over-length HBV genome into pUC18 (Takara, China) (Supplementary Fig. ). .. HDI plasmids were constructed by cloning ~1.3 fold over-length HBV genome into pUC18 (Takara, China) (Supplementary Fig. ).

Isolation:

Article Title: Hepatitis B virus persistence in mice reveals IL-21 and IL-33 as regulators of viral clearance
Article Snippet: B200 and B6 were isolated from patients of Huashan Hospital with informed consent obtained in compliance with requirements of Huashan Hospital Bioethics Committee (Permit No. KY2014-024). .. HDI plasmids were constructed by cloning ~1.3 fold over-length HBV genome into pUC18 (Takara, China) (Supplementary Fig. ).

Labeling:

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: For experiments using EBV gene-derived constructs, transfections were performed in 100 mm plates using calcium phosphate with 15 µg of total DNA (0.5 µg of pTRE2-Flag.BDLF1, 0.25 µg of pCI-Flag.EB2 and 0.5 µg of pTet-On or pTet-Off expression vectors (Clontech) and pUC18 up to 15 µg). .. For experiments using EBV gene-derived constructs, transfections were performed in 100 mm plates using calcium phosphate with 15 µg of total DNA (0.5 µg of pTRE2-Flag.BDLF1, 0.25 µg of pCI-Flag.EB2 and 0.5 µg of pTet-On or pTet-Off expression vectors (Clontech) and pUC18 up to 15 µg).

Purification:

Article Title: A Rice gid1 Suppressor Mutant Reveals That Gibberellin Is Not Always Required for Interaction between Its Receptor, GID1, and DELLA Proteins
Article Snippet: Both amplified fragments were purified and mixed to produce the template for the following PCR reaction, which was performed using Eco-AtGID1b/F and AtGID1b-Bam-Xho/R primers. .. The amplified first and second exons of each soybean gene were joined using restriction enzyme sites and cloned into pUC18 (TAKARA) at restriction enzyme sites shown in -(8) online.

Article Title: Fifty-Year Trend Towards Suppression of Wolbachia-Induced Male-Killing by Its Butterfly Host, Hypolimnas bolina
Article Snippet: The PCR products were purified using a MinElute Gel Extraction Kit (QIAGEN), Alkaline Phosphatase Exonuclease I (shrimp) (Takara), a Suprec-02 spin-filter (Takara), or a Takara DNA Fragment Purification Kit (Takara). .. For cloning, some PCR products were cloned into pUC118 (Takara) or pUC18 (Takara) vectors and then used to transform Escherichia coli JM 109 or DH5α competent cells.

Sequencing:

Article Title: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm
Article Snippet: The Xba I-Xba I fragment including the MR1 sequence was then subcloned into pBluescript II SK (+) (Stratagene). .. Additional clones used included a BLV infectious clone, pBLV-IF [ ]; a HTLV-1 infectious clone, pK30 [ ]; a HIV-1 infectious clone, pNL4-3 [ ]; a SIV infectious clone, SIVmac239/WT [ ]; a hybrid MMTV provirus plasmid [ ]; a M-MLV infectious clone, pL-4 [ ] and plasmids including pUC18 (Takara Bio Inc., Tokyo, Japan), pUC19 (Takara Bio Inc.), and pBR322 (Promega).

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: A fragment containing the full sequence of PhrdB promoter (426 bp) with a 5’ Eco RI site and a 3’ Nco I site was amplified by PCR from S . coelicolor M145 genomic DNA using primers H1/H2. .. The resulting PCR products were cloned separately into pUC18 (TaKaRa, Japan), and the products were subsequently excised from the resulting constructs using Nco I-Hin dIII and Eco RI-Nco I and then ligated with the Hin dIII-Eco RI fragment from pSPU241.

Article Title: High Prevalence of Plasmid-Mediated Quinolone Resistance Determinants qnr, aac(6?)-Ib-cr, and qepA among Ceftiofur-Resistant Enterobacteriaceae Isolates from Companion and Food-Producing Animals
Article Snippet: To determine the genetic environment of aac(6 ′ )-Ib-cr or the qnr genes, plasmid DNA was digested with EcoRI or HindIII, ligated to pUC18 (which is resistant to ampicillin; TaKaRa Bio, Otsu, Japan), and introduced into E. coli DH5α. .. To determine the genetic environment of aac(6 ′ )-Ib-cr or the qnr genes, plasmid DNA was digested with EcoRI or HindIII, ligated to pUC18 (which is resistant to ampicillin; TaKaRa Bio, Otsu, Japan), and introduced into E. coli DH5α.

Article Title: Characterization of Extended-Spectrum ?-Lactamase Genes Found among Escherichia coli Isolates from Duck and Environmental Samples Obtained on a Duck Farm
Article Snippet: To characterize the genetic environment of the bla CTX-M gene, plasmid DNA was digested with EcoRI, ligated into pUC18 (TaKaRa Biotechnology, Dalian, China), and introduced into E. coli DH5α. .. To characterize the genetic environment of the bla CTX-M gene, plasmid DNA was digested with EcoRI, ligated into pUC18 (TaKaRa Biotechnology, Dalian, China), and introduced into E. coli DH5α.

Article Title: Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli
Article Snippet: The full-length sequence of E. coli tesA was amplified and cloned into plasmid pUC18S. .. Plasmid pUC18S was created by replacing the Aat II/Eam 1105I fragment (containing the ampicillin-resistance gene) of pUC18 (Takara) with an Aat II/Eam 1105I fragment (containing the spectinomycin-resistance gene) amplified, by PCR, from pAPNC213 [ ].

Article Title: Fifty-Year Trend Towards Suppression of Wolbachia-Induced Male-Killing by Its Butterfly Host, Hypolimnas bolina
Article Snippet: Paragraph title: Sequencing ... For cloning, some PCR products were cloned into pUC118 (Takara) or pUC18 (Takara) vectors and then used to transform Escherichia coli JM 109 or DH5α competent cells.

FACS:

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: For experiments using EBV gene-derived constructs, transfections were performed in 100 mm plates using calcium phosphate with 15 µg of total DNA (0.5 µg of pTRE2-Flag.BDLF1, 0.25 µg of pCI-Flag.EB2 and 0.5 µg of pTet-On or pTet-Off expression vectors (Clontech) and pUC18 up to 15 µg). .. For the metabolic labeling experiments, transfection of HeLa cells with the EB2 expression plasmid was carried out in six-well plates using Lipofectamine 2000 (Invitrogen).

Mouse Assay:

Article Title: Effects of different interchain linkers on biological activity of an anti-prostate cancer single-chain bispecific antibody
Article Snippet: The pUC18 and pSectag2B plasmids, restriction endonucleases, and DNA ligase were all purchased from TaKaRa Bio Inc. (Tokyo, Japan); plasmids pUC18-γ-Sm ScFv and pUC18-CD3 ScFv were constructed and stored in our laboratory. .. The ELISA kit was purchased from Shanghai Ding Biological Technology Co., Ltd. (Shanghai, China).

Plasmid Preparation:

Article Title: Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis
Article Snippet: Escherichia coli DH5α (Transgen, China) was used for plasmid construction and propagation according to the manufacturer’s instructions. .. To functionally express ptTES1 in E. coli XL1-blue, the codon-optimized ptTES1 gene was synthesized and cloned in frame into pUC18 (Takara, China), generating the pUC18-phaeoTES1 plasmid. .. To overexpress ptTES1 in E. coli Rosetta (DE3), the codon-optimized ptTES1 gene was synthesized and cloned into pET28a (Novagen), generating the pET28a-phaeoTES1 plasmid.

Article Title: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm
Article Snippet: The Xba I-Xba I fragment including the MR1 sequence was then subcloned into pBluescript II SK (+) (Stratagene). .. Additional clones used included a BLV infectious clone, pBLV-IF [ ]; a HTLV-1 infectious clone, pK30 [ ]; a HIV-1 infectious clone, pNL4-3 [ ]; a SIV infectious clone, SIVmac239/WT [ ]; a hybrid MMTV provirus plasmid [ ]; a M-MLV infectious clone, pL-4 [ ] and plasmids including pUC18 (Takara Bio Inc., Tokyo, Japan), pUC19 (Takara Bio Inc.), and pBR322 (Promega). .. pBLV-LTR/SK and pBoLA-DRA/SK were digested with Sca I and purified using a Sephadex G-50 column (GE Healthcare Japan, Tokyo, Japan).

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The pHJK1 plasmid containing the PhrdB promoter, kanJ and kanK was constructed for overexpression. .. The resulting PCR products were cloned separately into pUC18 (TaKaRa, Japan), and the products were subsequently excised from the resulting constructs using Nco I-Hin dIII and Eco RI-Nco I and then ligated with the Hin dIII-Eco RI fragment from pSPU241.

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The plasmid pDLA302 containing kanN with an in-frame deletion was constructed as follows. .. N1 and N2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids using Xba I-EcoR I and Eco RI-Kpn I, respectively.

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: The plasmid pLDJ202 containing kanJ with in-frame deletion was constructed as follows. .. J1 and J2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids with Eco RI-Hin dIII and Eco RI-Bam HI, respectively.

Article Title: Molecular insights into replication initiation by Qβ replicase using ribosomal protein S1
Article Snippet: All structural figures were prepared by the PyMOL Molecular Graphics System (Schrödinger, LLC), and the protein–protein interfaces of the complex were analyzed with PISA ( ). .. The plasmid (pT7QB), bearing the T7 promoter and the DNA corresponding to the Qβ positive strand RNA in pUC18, was purchased from Takara, Japan. .. The positive strand Qβ RNA was prepared with a MEGAScript T7 Transcription Kit (Invitrogen), using the pT7QB plasmid linearized by Eco T22I.

Article Title: Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression
Article Snippet: The CmR gene cassette was PCR amplified from pUC18-Cm ( ) with primer pair CmRU/CmRD (all PCR primers are listed in ) and cloned into pUC18 (TaKaRa) to generate pUC18-CmR. .. Two homologous fragments flanking the deletion region were PCR amplified from bMON14272 using primer pairs pp78/83-U/pp78/83-D and pk1-U/pk1-D.

Article Title: High Prevalence of Plasmid-Mediated Quinolone Resistance Determinants qnr, aac(6?)-Ib-cr, and qepA among Ceftiofur-Resistant Enterobacteriaceae Isolates from Companion and Food-Producing Animals
Article Snippet: Plasmid sizes were estimated as described previously ( ). .. To determine the genetic environment of aac(6 ′ )-Ib-cr or the qnr genes, plasmid DNA was digested with EcoRI or HindIII, ligated to pUC18 (which is resistant to ampicillin; TaKaRa Bio, Otsu, Japan), and introduced into E. coli DH5α. .. Transformants were selected on tryptic soy agar plates containing ampicillin at 50 μg/ml and ciprofloxacin at 0.06 μg/ml.

Article Title: Characterization of Extended-Spectrum ?-Lactamase Genes Found among Escherichia coli Isolates from Duck and Environmental Samples Obtained on a Duck Farm
Article Snippet: The results were interpreted according to the criteria of Tenover et al. ( ). .. To characterize the genetic environment of the bla CTX-M gene, plasmid DNA was digested with EcoRI, ligated into pUC18 (TaKaRa Biotechnology, Dalian, China), and introduced into E. coli DH5α. .. Transformants were selected on LB agar plates containing 4 μg/ml cefotaxime.

Article Title: Pathway engineering of Propionibacterium jensenii for improved production of propionic acid
Article Snippet: P. jensenii ATCC 4868 and K. pneumoniae subsp. pneumoniae ATCC 12657 were purchased from the American Type Culture Collection (ATCC) and used as templates to amplify genes or as hosts for gene expression and deletion. .. E. coli JM110 (Stratagene, La Jolla, CA) was used for plasmid cloning and maintenance. pUC18 (TaKaRa, Dalian, China) was used as suicide plasmid to knock out genes in P. jensenii , and the shuttle vector pZGX04 was constructed in our previous study . pRS303 was maintained in our laboratory to amplify the hygromycin B resistance gene (hygB ). .. P. jensenii was cultured anaerobically at 32 °C in sodium lactate broth medium (10 g·L−1 yeast extract, 10 g·L−1 tryptic soy broth, and 10 g·L−1 sodium lactate), and E. coli and K. pneumoniae were grown at 37 °C in Luria-Bertani medium (10 g·L−1 NaCl, 10 g·L−1 peptone, and 5 g·L−1 yeast extract) with a shaking speed of 220 rpm.

Article Title: Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1
Article Snippet: The used strains and vectors were of E. coli DH5α, E. coli BL21 (DE3) (Tiangen), pUC18 (Takara), and pET30a(+) (Novagen). .. Luria–Bertani medium that containing 10.0 g·L−1 of tryptone, 5.0 g·L−1 of yeast extract, and 10.0 g·L−1 of NaCl was used for recombinant protein expression.

Article Title: Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2
Article Snippet: For experiments using EBV gene-derived constructs, transfections were performed in 100 mm plates using calcium phosphate with 15 µg of total DNA (0.5 µg of pTRE2-Flag.BDLF1, 0.25 µg of pCI-Flag.EB2 and 0.5 µg of pTet-On or pTet-Off expression vectors (Clontech) and pUC18 up to 15 µg). .. For experiments using EBV gene-derived constructs, transfections were performed in 100 mm plates using calcium phosphate with 15 µg of total DNA (0.5 µg of pTRE2-Flag.BDLF1, 0.25 µg of pCI-Flag.EB2 and 0.5 µg of pTet-On or pTet-Off expression vectors (Clontech) and pUC18 up to 15 µg).

Article Title: Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies
Article Snippet: The amplified 5′ and 3′ polyhedrin segments were analyzed by agarose gel electrophoresis and then recovered from the gel by use of a gel extraction kit (Omega Bio-Tek, Norcross, GA, USA), and they were digested with NheI/SphI and EcoRI/XbaI, respectively, and then ligated into pMD18T and pUC18 (TaKaRa), respectively. .. The resulting constructs are here called pMD18T-phN110, pMD18T-phN150, pMD18T-phN170, and pMD18T-phN204 (pMD18T constructs) and pUC18-phC135, pUC18-phC95, pUC18-phC75, and pUC18-phC41 (pUC18 constructs).

Article Title: A Rice gid1 Suppressor Mutant Reveals That Gibberellin Is Not Always Required for Interaction between Its Receptor, GID1, and DELLA Proteins
Article Snippet: Paragraph title: Plasmid Construction ... The amplified first and second exons of each soybean gene were joined using restriction enzyme sites and cloned into pUC18 (TAKARA) at restriction enzyme sites shown in -(8) online.

Article Title: Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both Bacillus subtilis and Escherichia coli
Article Snippet: The full-length sequence of E. coli tesA was amplified and cloned into plasmid pUC18S. .. Plasmid pUC18S was created by replacing the Aat II/Eam 1105I fragment (containing the ampicillin-resistance gene) of pUC18 (Takara) with an Aat II/Eam 1105I fragment (containing the spectinomycin-resistance gene) amplified, by PCR, from pAPNC213 [ ]. .. B. subtilis strains were grown in Luria-Bertani (LB) medium, or DSM, supplemented with chloramphenicol (5 μg/ml), kanamycin (5 μg/ml), or erythromycin (0.5 μg/ml), as appropriate.E. coli strains were grown in LB or M9 minimal media, supplemented with ampicillin (50 μg/ml), chloramphenicol (5 μg/ml), spectinomycin (50 μg/ml), or kanamycin (25 μg/ml), as appropriate.

Article Title: Improved Adsorption of an Enterococcus faecalis Bacteriophage ?EF24C with a Spontaneous Point Mutation
Article Snippet: E. faecalis phage ΦEF24C is described elsewhere – . pUC18 and pCold II plasmids were purchased from Takara Bio (Kyoto, Japan) for the purposes of cloning and protein overexpression, respectively. .. E. faecalis phage ΦEF24C is described elsewhere – . pUC18 and pCold II plasmids were purchased from Takara Bio (Kyoto, Japan) for the purposes of cloning and protein overexpression, respectively.

Agarose Gel Electrophoresis:

Article Title: Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies
Article Snippet: The gp37 (CpGV orf13 ) ( ) and en4 (nucleotides [nt] 616 to 1499 of AgseGV enhancin [GenBank accession no. ]) sequences were PCR amplified from the genomic DNA of the corresponding virus by using the gp37-F/gp37-R or en4-F/en4-R primer pair ( ). .. The amplified 5′ and 3′ polyhedrin segments were analyzed by agarose gel electrophoresis and then recovered from the gel by use of a gel extraction kit (Omega Bio-Tek, Norcross, GA, USA), and they were digested with NheI/SphI and EcoRI/XbaI, respectively, and then ligated into pMD18T and pUC18 (TaKaRa), respectively. .. The resulting constructs are here called pMD18T-phN110, pMD18T-phN150, pMD18T-phN170, and pMD18T-phN204 (pMD18T constructs) and pUC18-phC135, pUC18-phC95, pUC18-phC75, and pUC18-phC41 (pUC18 constructs).

In Vitro:

Article Title: Molecular insights into replication initiation by Qβ replicase using ribosomal protein S1
Article Snippet: Paragraph title: In vitro negative strand synthesis by Qβ replicase variants ... The plasmid (pT7QB), bearing the T7 promoter and the DNA corresponding to the Qβ positive strand RNA in pUC18, was purchased from Takara, Japan.

Chromosome Walking:

Article Title: High Prevalence of Plasmid-Mediated Quinolone Resistance Determinants qnr, aac(6?)-Ib-cr, and qepA among Ceftiofur-Resistant Enterobacteriaceae Isolates from Companion and Food-Producing Animals
Article Snippet: To determine the genetic environment of aac(6 ′ )-Ib-cr or the qnr genes, plasmid DNA was digested with EcoRI or HindIII, ligated to pUC18 (which is resistant to ampicillin; TaKaRa Bio, Otsu, Japan), and introduced into E. coli DH5α. .. To determine the genetic environment of aac(6 ′ )-Ib-cr or the qnr genes, plasmid DNA was digested with EcoRI or HindIII, ligated to pUC18 (which is resistant to ampicillin; TaKaRa Bio, Otsu, Japan), and introduced into E. coli DH5α.

Article Title: Characterization of Extended-Spectrum ?-Lactamase Genes Found among Escherichia coli Isolates from Duck and Environmental Samples Obtained on a Duck Farm
Article Snippet: To characterize the genetic environment of the bla CTX-M gene, plasmid DNA was digested with EcoRI, ligated into pUC18 (TaKaRa Biotechnology, Dalian, China), and introduced into E. coli DH5α. .. To characterize the genetic environment of the bla CTX-M gene, plasmid DNA was digested with EcoRI, ligated into pUC18 (TaKaRa Biotechnology, Dalian, China), and introduced into E. coli DH5α.

Knock-Out:

Article Title: Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression
Article Snippet: Thus, to generate a complete pk1 knockout virus and to avoid any impact on pp78/83 expression, a 1,575-bp region containing part of the pp78/83 and pk1 coding regions of bMON14272 (nt 5,936 to 7,510) was replaced with a 1,038-bp chloramphenicol resistance ( CmR ) gene cassette, and then a copy of pp78/83 was inserted into the mini- att Tn 7 site via site-specific Tn7 transposition ( ). .. The CmR gene cassette was PCR amplified from pUC18-Cm ( ) with primer pair CmRU/CmRD (all PCR primers are listed in ) and cloned into pUC18 (TaKaRa) to generate pUC18-CmR.

Article Title: Pathway engineering of Propionibacterium jensenii for improved production of propionic acid
Article Snippet: P. jensenii ATCC 4868 and K. pneumoniae subsp. pneumoniae ATCC 12657 were purchased from the American Type Culture Collection (ATCC) and used as templates to amplify genes or as hosts for gene expression and deletion. .. E. coli JM110 (Stratagene, La Jolla, CA) was used for plasmid cloning and maintenance. pUC18 (TaKaRa, Dalian, China) was used as suicide plasmid to knock out genes in P. jensenii , and the shuttle vector pZGX04 was constructed in our previous study . pRS303 was maintained in our laboratory to amplify the hygromycin B resistance gene (hygB ). .. P. jensenii was cultured anaerobically at 32 °C in sodium lactate broth medium (10 g·L−1 yeast extract, 10 g·L−1 tryptic soy broth, and 10 g·L−1 sodium lactate), and E. coli and K. pneumoniae were grown at 37 °C in Luria-Bertani medium (10 g·L−1 NaCl, 10 g·L−1 peptone, and 5 g·L−1 yeast extract) with a shaking speed of 220 rpm.

Produced:

Article Title: A Rice gid1 Suppressor Mutant Reveals That Gibberellin Is Not Always Required for Interaction between Its Receptor, GID1, and DELLA Proteins
Article Snippet: GID1 cDNAs from various species for Y2H assays were produced by PCR or synthesized based on the database sequences of mRNA or predicted coding sequences. cDNAs of grape ( Vitis vinifera ) GID1a , grape GID1b , and Brassica napus GID1 were synthesized with Eco RI- Bam HI, Eco RI- Bam HI and Eco RI- Sma I sites, respectively (GenScript), and cloned into pGBKT7. cDNAs of soybean GID1a-1 , a-2 , b-1 , b-2 , and b-3 were produced as follows. .. The amplified first and second exons of each soybean gene were joined using restriction enzyme sites and cloned into pUC18 (TAKARA) at restriction enzyme sites shown in -(8) online.

Concentration Assay:

Article Title: Modulation of kanamycin B and kanamycin A biosynthesis in Streptomyces kanamyceticus via metabolic engineering
Article Snippet: N1 and N2 were cloned separately into pUC18 (TaKaRa, Japan) and then excised from the resulting plasmids using Xba I-EcoR I and Eco RI-Kpn I, respectively. .. First, the disruption plasmid pDLA302 was introduced into E . coli ET12567/pUZ8002 via the CaCl2 method and then into S . kanamyceticus CG305 by conjugational transfer [ ].

Gel Extraction:

Article Title: Improving Baculovirus Infectivity by Efficiently Embedding Enhancing Factors into Occlusion Bodies
Article Snippet: The gp37 (CpGV orf13 ) ( ) and en4 (nucleotides [nt] 616 to 1499 of AgseGV enhancin [GenBank accession no. ]) sequences were PCR amplified from the genomic DNA of the corresponding virus by using the gp37-F/gp37-R or en4-F/en4-R primer pair ( ). .. The amplified 5′ and 3′ polyhedrin segments were analyzed by agarose gel electrophoresis and then recovered from the gel by use of a gel extraction kit (Omega Bio-Tek, Norcross, GA, USA), and they were digested with NheI/SphI and EcoRI/XbaI, respectively, and then ligated into pMD18T and pUC18 (TaKaRa), respectively. .. The resulting constructs are here called pMD18T-phN110, pMD18T-phN150, pMD18T-phN170, and pMD18T-phN204 (pMD18T constructs) and pUC18-phC135, pUC18-phC95, pUC18-phC75, and pUC18-phC41 (pUC18 constructs).

Article Title: Fifty-Year Trend Towards Suppression of Wolbachia-Induced Male-Killing by Its Butterfly Host, Hypolimnas bolina
Article Snippet: The PCR products were purified using a MinElute Gel Extraction Kit (QIAGEN), Alkaline Phosphatase Exonuclease I (shrimp) (Takara), a Suprec-02 spin-filter (Takara), or a Takara DNA Fragment Purification Kit (Takara). .. For cloning, some PCR products were cloned into pUC118 (Takara) or pUC18 (Takara) vectors and then used to transform Escherichia coli JM 109 or DH5α competent cells.

Variant Assay:

Article Title: Molecular insights into replication initiation by Qβ replicase using ribosomal protein S1
Article Snippet: The plasmid (pT7QB), bearing the T7 promoter and the DNA corresponding to the Qβ positive strand RNA in pUC18, was purchased from Takara, Japan. .. The plasmid (pT7QB), bearing the T7 promoter and the DNA corresponding to the Qβ positive strand RNA in pUC18, was purchased from Takara, Japan.

Homologous Recombination:

Article Title: Posttranslational Modifications of Baculovirus Protamine-Like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression
Article Snippet: The bacmid bMON14272 (Invitrogen), which contains the genome of AcMNPV (strain E2) , was used to generate a pk1 -null virus by homologous recombination in Escherichia coli as previously described ( ). .. The CmR gene cassette was PCR amplified from pUC18-Cm ( ) with primer pair CmRU/CmRD (all PCR primers are listed in ) and cloned into pUC18 (TaKaRa) to generate pUC18-CmR.

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  • 84
    TaKaRa puc18 plasmid
    Effects of different nanoparticles on the transformablities of plasmid DNA. Notes: DNA qualities of plasmids <t>pUC18</t> incubated with nano-TiO 2 (1.5 mg/mL), fullerenes (1.5 mg/mL), MAA–QDs (3.6 μmol/L) and OPA-QDs (3 μmol/L) for 2 hours at 4°C in the dark were tested by transformation with Escherichia coli strain DH5α. There is no significant difference between the incubated and non-incubated plasmids in transformation activity when plasmids were incubated with nano-TiO 2 , fullerenes, and OPA-QDs ( P > 0.05), respectively. Abbreviations: MAA-QDs, mercaptoacetic acid-coated quantum dots; OPA-QDs, octylamine-modified polyacrylic acid-coated quantum dots.
    Puc18 Plasmid, supplied by TaKaRa, used in various techniques. Bioz Stars score: 84/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/puc18 plasmid/product/TaKaRa
    Average 84 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    puc18 plasmid - by Bioz Stars, 2019-10
    84/100 stars
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    77
    TaKaRa puc18 template dna
    Representative ultraviolet images from ethidium bromide-stained PAGE gels of the 110 nt PCR product derived from <t>pUC18</t> (amplifying region I) and the C5-modified nucleoside triphosphates. ( A–C ) The reaction mixtures containing dATP, dGTP, dCTP and T AL (lane 3), T AC (lane 4), T AF (lane 5), T PA (lane 6), T PN (lane 7), T PR (lane 8), T A2 (lane 11), T A4 (lane 12), T A6 (lane 13), T G6 (lane 14), T ME (lane 15), T CN (lane 16), or T DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures do not contain natural TTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and dCTP; lanes 1 and 9). ( D–F ) Reaction mixtures containing dATP, dGTP, TTP and C AL (lane 3), C AC (lane 4), C AF (lane 5), C PA (lane 6), C PN (lane 7), C PR (lane 8), C A2 (lane 11), C A4 (lane 12), C A6 (lane 13), C G6 (lane 14), C ME (lane 15), C CN (lane 16) or C DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures did not contain natural dCTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and TTP; lanes 1 and 9). The thermostable <t>DNA</t> polymerases used were Taq (A, D), Vent(exo-) (B and E), and KOD Dash (C and F).
    Puc18 Template Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/puc18 template dna/product/TaKaRa
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    puc18 template dna - by Bioz Stars, 2019-10
    77/100 stars
      Buy from Supplier

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    Effects of different nanoparticles on the transformablities of plasmid DNA. Notes: DNA qualities of plasmids pUC18 incubated with nano-TiO 2 (1.5 mg/mL), fullerenes (1.5 mg/mL), MAA–QDs (3.6 μmol/L) and OPA-QDs (3 μmol/L) for 2 hours at 4°C in the dark were tested by transformation with Escherichia coli strain DH5α. There is no significant difference between the incubated and non-incubated plasmids in transformation activity when plasmids were incubated with nano-TiO 2 , fullerenes, and OPA-QDs ( P > 0.05), respectively. Abbreviations: MAA-QDs, mercaptoacetic acid-coated quantum dots; OPA-QDs, octylamine-modified polyacrylic acid-coated quantum dots.

    Journal: International Journal of Nanomedicine

    Article Title: The cadmium-mercaptoacetic acid complex contributes to the genotoxicity of mercaptoacetic acid-coated CdSe-core quantum dots

    doi: 10.2147/IJN.S32029

    Figure Lengend Snippet: Effects of different nanoparticles on the transformablities of plasmid DNA. Notes: DNA qualities of plasmids pUC18 incubated with nano-TiO 2 (1.5 mg/mL), fullerenes (1.5 mg/mL), MAA–QDs (3.6 μmol/L) and OPA-QDs (3 μmol/L) for 2 hours at 4°C in the dark were tested by transformation with Escherichia coli strain DH5α. There is no significant difference between the incubated and non-incubated plasmids in transformation activity when plasmids were incubated with nano-TiO 2 , fullerenes, and OPA-QDs ( P > 0.05), respectively. Abbreviations: MAA-QDs, mercaptoacetic acid-coated quantum dots; OPA-QDs, octylamine-modified polyacrylic acid-coated quantum dots.

    Article Snippet: The pUC18 plasmid ( > 70% supercoiled) was purchased from TaKaRa Biotech.

    Techniques: Plasmid Preparation, Incubation, Transformation Assay, Activity Assay, Modification

    Effects of MAA-coated CdSe QDs on the plasmid DNA. ( A ) Electrophoresis in 1% agarose gel of pUC18 DNA (150 ng per sample) incubated for 2 hours at 4°C in the dark with QDs. Lane 1: pUC18 DNA only; lanes 2–6: pUC18 DNA incubated with different concentrations of QDs (3.6, 1.2, 0.4, 0.13, 0.043 μmol/L). ( B ) DNA quality of plasmids pUC18 incubated with different concentrations of QDs (3.6, 1.2, 0.4, 0.13, 0.043 μmol/L) for 2 hours at 4°C in the dark was tested by transformation with Escherichia coli strain DH5α. ( C ) Scanning densitometry results of three replicate experiments for each sample, with the error bars representing the standard deviations. Abbreviations: OC, opened circular; CCC, covalently closed circular; MAA, mercaptoacetic acid; QDs, quantum dots.

    Journal: International Journal of Nanomedicine

    Article Title: The cadmium-mercaptoacetic acid complex contributes to the genotoxicity of mercaptoacetic acid-coated CdSe-core quantum dots

    doi: 10.2147/IJN.S32029

    Figure Lengend Snippet: Effects of MAA-coated CdSe QDs on the plasmid DNA. ( A ) Electrophoresis in 1% agarose gel of pUC18 DNA (150 ng per sample) incubated for 2 hours at 4°C in the dark with QDs. Lane 1: pUC18 DNA only; lanes 2–6: pUC18 DNA incubated with different concentrations of QDs (3.6, 1.2, 0.4, 0.13, 0.043 μmol/L). ( B ) DNA quality of plasmids pUC18 incubated with different concentrations of QDs (3.6, 1.2, 0.4, 0.13, 0.043 μmol/L) for 2 hours at 4°C in the dark was tested by transformation with Escherichia coli strain DH5α. ( C ) Scanning densitometry results of three replicate experiments for each sample, with the error bars representing the standard deviations. Abbreviations: OC, opened circular; CCC, covalently closed circular; MAA, mercaptoacetic acid; QDs, quantum dots.

    Article Snippet: The pUC18 plasmid ( > 70% supercoiled) was purchased from TaKaRa Biotech.

    Techniques: Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Incubation, Transformation Assay, Countercurrent Chromatography

    CD spectra of pUC18 DNA. Notes: The interactions of pUC18 DNA with MAA and Cd–MAA were at a ratio of compound:DNA = 0.4. All the spectra were recorded in Tris-HCl buffer, pH 7.0. Abbreviations: CD, circular dichroism; MAA, mercaptoacetic acid; Cd-MAA, cadmium-mercaptoacetic acid.

    Journal: International Journal of Nanomedicine

    Article Title: The cadmium-mercaptoacetic acid complex contributes to the genotoxicity of mercaptoacetic acid-coated CdSe-core quantum dots

    doi: 10.2147/IJN.S32029

    Figure Lengend Snippet: CD spectra of pUC18 DNA. Notes: The interactions of pUC18 DNA with MAA and Cd–MAA were at a ratio of compound:DNA = 0.4. All the spectra were recorded in Tris-HCl buffer, pH 7.0. Abbreviations: CD, circular dichroism; MAA, mercaptoacetic acid; Cd-MAA, cadmium-mercaptoacetic acid.

    Article Snippet: The pUC18 plasmid ( > 70% supercoiled) was purchased from TaKaRa Biotech.

    Techniques:

    Effect of Cd 2+ or MAA on the configuration of plasmid DNA. Notes: Electrophoresis in 1% agarose gel of pUC18 DNA (150 ng per sample) incubated for 12 hours at 4°C in the dark with increasing concentrations of Cd 2+ or with increasing concentrations of MAA. Lane 1: pUC18 DNA only; lane 2: pUC18 DNA digested by Hin d III; lanes 3–6: pUC18 DNA incubated with 0.5, 5, 50, 500 μmol/L Cd ions; lanes 7–10: pUC18 DNA incubated with 0.05, 0.5, 5, 50 mmol/L MAA. DNA smear caused by MAA was observed in lanes 8–10, and the pUC18 plasmid DNA in lane 10 was completely degraded by MAA. Abbreviations: OC, opened circular; CCC, covalently closed circular; MAA, mercaptoacetic acid.

    Journal: International Journal of Nanomedicine

    Article Title: The cadmium-mercaptoacetic acid complex contributes to the genotoxicity of mercaptoacetic acid-coated CdSe-core quantum dots

    doi: 10.2147/IJN.S32029

    Figure Lengend Snippet: Effect of Cd 2+ or MAA on the configuration of plasmid DNA. Notes: Electrophoresis in 1% agarose gel of pUC18 DNA (150 ng per sample) incubated for 12 hours at 4°C in the dark with increasing concentrations of Cd 2+ or with increasing concentrations of MAA. Lane 1: pUC18 DNA only; lane 2: pUC18 DNA digested by Hin d III; lanes 3–6: pUC18 DNA incubated with 0.5, 5, 50, 500 μmol/L Cd ions; lanes 7–10: pUC18 DNA incubated with 0.05, 0.5, 5, 50 mmol/L MAA. DNA smear caused by MAA was observed in lanes 8–10, and the pUC18 plasmid DNA in lane 10 was completely degraded by MAA. Abbreviations: OC, opened circular; CCC, covalently closed circular; MAA, mercaptoacetic acid.

    Article Snippet: The pUC18 plasmid ( > 70% supercoiled) was purchased from TaKaRa Biotech.

    Techniques: Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Incubation, Countercurrent Chromatography

    Co-effect of Cd 2+ and MAA on the configuration of plasmid DNA. ( A ) Electrophoresis in 1% agarose gel of pUC18 DNA (150 ng per sample) incubated with Cd 2+ in the presence of increasing MAA concentrations for 12 hours at 4°C in the dark. Lane 1: pUC18 DNA only; lane 2: pUC18 DNA digested by Hin d III; lanes 3–9: pUC18 DNA incubated with mixtures of Cd 2+ (500 μmol/L) and 0, 5, 10, 15, 25, 35, 45 mmol/L MAA. ( B ) Scanning densitometry results of three replicate experiments for each sample, with the error bars representing the standard deviations. Abbreviations: OC, opened circular; CCC, covalently closed circular; MAA, mercaptoacetic acid.

    Journal: International Journal of Nanomedicine

    Article Title: The cadmium-mercaptoacetic acid complex contributes to the genotoxicity of mercaptoacetic acid-coated CdSe-core quantum dots

    doi: 10.2147/IJN.S32029

    Figure Lengend Snippet: Co-effect of Cd 2+ and MAA on the configuration of plasmid DNA. ( A ) Electrophoresis in 1% agarose gel of pUC18 DNA (150 ng per sample) incubated with Cd 2+ in the presence of increasing MAA concentrations for 12 hours at 4°C in the dark. Lane 1: pUC18 DNA only; lane 2: pUC18 DNA digested by Hin d III; lanes 3–9: pUC18 DNA incubated with mixtures of Cd 2+ (500 μmol/L) and 0, 5, 10, 15, 25, 35, 45 mmol/L MAA. ( B ) Scanning densitometry results of three replicate experiments for each sample, with the error bars representing the standard deviations. Abbreviations: OC, opened circular; CCC, covalently closed circular; MAA, mercaptoacetic acid.

    Article Snippet: The pUC18 plasmid ( > 70% supercoiled) was purchased from TaKaRa Biotech.

    Techniques: Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Incubation, Countercurrent Chromatography

    Schematic diagrams of the plasmids. (A) pUC18-HVT-TK. The region from 45,700 to 48,967 nucleotides (nts) of the herpesvirus of turkeys (HVT) FC126 strain genome was cloned into pUC18. (B) pUC18-HVT-TK- Sfi I. The Sfi I recognition site was introduced between nts 47,316 and 47,317 of the FC126 genome, and the 45,700–48,967 region was cloned into pUC18. (C) pUC18-HVT-TK-I- Sce I- Sfi I. A 50-bp duplication sequence (nts 47,317–47,366 of the FC126 genome) and the I- Sce I recognition site were inserted before the Sfi I site. Dashed lines show homologous sequences. (D) pUC18-HVT-BAC. LoxP , eGFP, mini-F and chloramphenicol resistance cassette sequences were inserted into the Sfi I site of pUC18-HVT-TK-I- Sce I- Sfi I. Dashed lines show homologous sequences. Cm R indicates the chloramphenicol resistance gene.

    Journal: The Journal of Veterinary Medical Science

    Article Title: Removal of Inserted BAC after linearizatiON (RIBON)—a novel strategy to excise the mini-F sequences from viral BAC vectors

    doi: 10.1292/jvms.16-0038

    Figure Lengend Snippet: Schematic diagrams of the plasmids. (A) pUC18-HVT-TK. The region from 45,700 to 48,967 nucleotides (nts) of the herpesvirus of turkeys (HVT) FC126 strain genome was cloned into pUC18. (B) pUC18-HVT-TK- Sfi I. The Sfi I recognition site was introduced between nts 47,316 and 47,317 of the FC126 genome, and the 45,700–48,967 region was cloned into pUC18. (C) pUC18-HVT-TK-I- Sce I- Sfi I. A 50-bp duplication sequence (nts 47,317–47,366 of the FC126 genome) and the I- Sce I recognition site were inserted before the Sfi I site. Dashed lines show homologous sequences. (D) pUC18-HVT-BAC. LoxP , eGFP, mini-F and chloramphenicol resistance cassette sequences were inserted into the Sfi I site of pUC18-HVT-TK-I- Sce I- Sfi I. Dashed lines show homologous sequences. Cm R indicates the chloramphenicol resistance gene.

    Article Snippet: The amplified fragment was digested withSal I and Sac I and cloned into the pUC18 vector (Takara, Otsu, Japan), resulting in pUC18-HVT-TK ( ).

    Techniques: Clone Assay, Sequencing, BAC Assay

    Plaques produced by reconstituted HVT- Sfi I in chicken embryo fibroblasts. Cells were transfected with linearized pHVT-BAC and pUC18-HVT-TK- Sfi I and analyzed for plaque formation and eGFP expression five days after transfection. Left panels, eGFP fluorescence; right panels, bright field microscopy. Scale bars, 50 µ m.

    Journal: The Journal of Veterinary Medical Science

    Article Title: Removal of Inserted BAC after linearizatiON (RIBON)—a novel strategy to excise the mini-F sequences from viral BAC vectors

    doi: 10.1292/jvms.16-0038

    Figure Lengend Snippet: Plaques produced by reconstituted HVT- Sfi I in chicken embryo fibroblasts. Cells were transfected with linearized pHVT-BAC and pUC18-HVT-TK- Sfi I and analyzed for plaque formation and eGFP expression five days after transfection. Left panels, eGFP fluorescence; right panels, bright field microscopy. Scale bars, 50 µ m.

    Article Snippet: The amplified fragment was digested withSal I and Sac I and cloned into the pUC18 vector (Takara, Otsu, Japan), resulting in pUC18-HVT-TK ( ).

    Techniques: Produced, Transfection, BAC Assay, Expressing, Fluorescence, Microscopy

    Representative ultraviolet images from ethidium bromide-stained PAGE gels of the 110 nt PCR product derived from pUC18 (amplifying region I) and the C5-modified nucleoside triphosphates. ( A–C ) The reaction mixtures containing dATP, dGTP, dCTP and T AL (lane 3), T AC (lane 4), T AF (lane 5), T PA (lane 6), T PN (lane 7), T PR (lane 8), T A2 (lane 11), T A4 (lane 12), T A6 (lane 13), T G6 (lane 14), T ME (lane 15), T CN (lane 16), or T DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures do not contain natural TTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and dCTP; lanes 1 and 9). ( D–F ) Reaction mixtures containing dATP, dGTP, TTP and C AL (lane 3), C AC (lane 4), C AF (lane 5), C PA (lane 6), C PN (lane 7), C PR (lane 8), C A2 (lane 11), C A4 (lane 12), C A6 (lane 13), C G6 (lane 14), C ME (lane 15), C CN (lane 16) or C DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures did not contain natural dCTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and TTP; lanes 1 and 9). The thermostable DNA polymerases used were Taq (A, D), Vent(exo-) (B and E), and KOD Dash (C and F).

    Journal: Nucleic Acids Research

    Article Title: Systematic characterization of 2?-deoxynucleoside- 5?-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA

    doi: 10.1093/nar/gkl637

    Figure Lengend Snippet: Representative ultraviolet images from ethidium bromide-stained PAGE gels of the 110 nt PCR product derived from pUC18 (amplifying region I) and the C5-modified nucleoside triphosphates. ( A–C ) The reaction mixtures containing dATP, dGTP, dCTP and T AL (lane 3), T AC (lane 4), T AF (lane 5), T PA (lane 6), T PN (lane 7), T PR (lane 8), T A2 (lane 11), T A4 (lane 12), T A6 (lane 13), T G6 (lane 14), T ME (lane 15), T CN (lane 16), or T DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures do not contain natural TTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and dCTP; lanes 1 and 9). ( D–F ) Reaction mixtures containing dATP, dGTP, TTP and C AL (lane 3), C AC (lane 4), C AF (lane 5), C PA (lane 6), C PN (lane 7), C PR (lane 8), C A2 (lane 11), C A4 (lane 12), C A6 (lane 13), C G6 (lane 14), C ME (lane 15), C CN (lane 16) or C DH (lane 17) were separated by electrophoresis on denaturing PAGE. Except for the positive control, the reaction mixtures did not contain natural dCTP. The PCR product was generated by the positive control (reaction mixture containing dATP, dGTP, dCTP and TTP; lanes 2 and 10) but not by the negative control (reaction mixture containing dATP, dGTP and TTP; lanes 1 and 9). The thermostable DNA polymerases used were Taq (A, D), Vent(exo-) (B and E), and KOD Dash (C and F).

    Article Snippet: Primers 1F, 1R, 2F and 2R were purchased from JBioS, and pUC18 template DNA was obtained from TaKaRa Biomedicals.

    Techniques: Staining, Polyacrylamide Gel Electrophoresis, Polymerase Chain Reaction, Derivative Assay, Modification, Electrophoresis, Positive Control, Generated, Negative Control

    Sequences of amplifying regions of the pUC18 template DNA. Primer sequences are underlined.

    Journal: Nucleic Acids Research

    Article Title: Systematic characterization of 2?-deoxynucleoside- 5?-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA

    doi: 10.1093/nar/gkl637

    Figure Lengend Snippet: Sequences of amplifying regions of the pUC18 template DNA. Primer sequences are underlined.

    Article Snippet: Primers 1F, 1R, 2F and 2R were purchased from JBioS, and pUC18 template DNA was obtained from TaKaRa Biomedicals.

    Techniques: