Structured Review

Boehringer Mannheim expand high fidelity pcr system
<t>PCR</t> amplification of fnr region from different <t>MG1655</t> isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.
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Images

1) Product Images from "Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression"

Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.18.5611-5626.2003

PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.
Figure Legend Snippet: PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

Techniques Used: Polymerase Chain Reaction, Amplification

2) Product Images from "The R3 Region, One of Three Major Repetitive Regions of Human Herpesvirus 6, Is a Strong Enhancer of Immediate-Early Gene U95"

Article Title: The R3 Region, One of Three Major Repetitive Regions of Human Herpesvirus 6, Is a Strong Enhancer of Immediate-Early Gene U95

Journal: Journal of Virology

doi: 10.1128/JVI.75.21.10149-10160.2001

Effect of repetition of the R3 unit on U95 promoter activity. (A) Schematic representation of PCR-generated R3 units containing NF-κB(R3) or NF-κB(TT) (termed R3-A and R3-B, respectively). (B) The left map represents the copy numbers of each R3 unit inserted immediately upstream of U95P-186. MT-4 cells were transfected with each construct and harvested 16 h later, and luciferase activities were measured. The right panel indicates the mean fold activities relative to that of pU95P-186-transfected cells in three independent experiments. The luciferase activities were normalized as described in Materials and Methods, and error bars plot the standard deviation for each set of triplicate samples.
Figure Legend Snippet: Effect of repetition of the R3 unit on U95 promoter activity. (A) Schematic representation of PCR-generated R3 units containing NF-κB(R3) or NF-κB(TT) (termed R3-A and R3-B, respectively). (B) The left map represents the copy numbers of each R3 unit inserted immediately upstream of U95P-186. MT-4 cells were transfected with each construct and harvested 16 h later, and luciferase activities were measured. The right panel indicates the mean fold activities relative to that of pU95P-186-transfected cells in three independent experiments. The luciferase activities were normalized as described in Materials and Methods, and error bars plot the standard deviation for each set of triplicate samples.

Techniques Used: Activity Assay, Polymerase Chain Reaction, Generated, Transfection, Construct, Luciferase, Standard Deviation

RT-PCR analyses for the transcripts of U95, IE1, Pol, gH, and EF 1α in HST-infected CBMCs treated with CHX and PFA. Total RNAs prepared from mock-infected and HST-infected cells, which had been treated with CHX for 24 h or PFA for 24 h or left untreated, were used. The U95 band was detectable in the presence of CHX and PFA, as well as the IE1 band, while the bands of Pol and gH could not be detected in the presence of CHX. EF1α, the cellular endogenous gene, was transcribed under all conditions.
Figure Legend Snippet: RT-PCR analyses for the transcripts of U95, IE1, Pol, gH, and EF 1α in HST-infected CBMCs treated with CHX and PFA. Total RNAs prepared from mock-infected and HST-infected cells, which had been treated with CHX for 24 h or PFA for 24 h or left untreated, were used. The U95 band was detectable in the presence of CHX and PFA, as well as the IE1 band, while the bands of Pol and gH could not be detected in the presence of CHX. EF1α, the cellular endogenous gene, was transcribed under all conditions.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Infection

3) Product Images from "Expression of a Gene for a Porin-Like Protein of the OmpA Family from Mycobacterium tuberculosis H37Rv"

Article Title: Expression of a Gene for a Porin-Like Protein of the OmpA Family from Mycobacterium tuberculosis H37Rv

Journal: Journal of Bacteriology

doi:

Expression of the gene for OmpATb in growing M. tuberculosis . Shown are gels scanned with a Leaf Lumina camera; images were processed on an Apple computer, using Adobe Photoshop version 3 and Macromedia Freehand version 5.5. (a) OmpATb and protein extracted from M. tuberculosis H37Rv were separated in SDS–10% polyacrylamide gels, blotted onto Immobilon-P membranes, and then detected with rabbit antiserum to truncated OmpATb. Lanes: 1, purified OmpATb; 2, protein from M. tuberculosis . The positions of marker proteins (not visible on the photograph) are indicated (in kilodaltons). Sample sizes were 50 ng for recombinant OmpATb and 100 μg for M. tuberculosis extract. (b) Gel of RT-PCR product obtained from mRNA of M. tuberculosis as described in Materials and Methods. Lanes 2 to 5 show products of PCRs with primers 1 and 3 and templates as follows: lane 2, reverse-transcribed M. tuberculosis RNA; lane 3, as for lane 2, but reverse transcriptase was omitted from the RT reaction mixture; lane 4, M. tuberculosis DNA; and lane 5, no template. Lanes 1 and 6 contain 100-bp ladder DNA (GIBCO-BRL; Life Technologies Ltd, Paisley, United Kingdom).
Figure Legend Snippet: Expression of the gene for OmpATb in growing M. tuberculosis . Shown are gels scanned with a Leaf Lumina camera; images were processed on an Apple computer, using Adobe Photoshop version 3 and Macromedia Freehand version 5.5. (a) OmpATb and protein extracted from M. tuberculosis H37Rv were separated in SDS–10% polyacrylamide gels, blotted onto Immobilon-P membranes, and then detected with rabbit antiserum to truncated OmpATb. Lanes: 1, purified OmpATb; 2, protein from M. tuberculosis . The positions of marker proteins (not visible on the photograph) are indicated (in kilodaltons). Sample sizes were 50 ng for recombinant OmpATb and 100 μg for M. tuberculosis extract. (b) Gel of RT-PCR product obtained from mRNA of M. tuberculosis as described in Materials and Methods. Lanes 2 to 5 show products of PCRs with primers 1 and 3 and templates as follows: lane 2, reverse-transcribed M. tuberculosis RNA; lane 3, as for lane 2, but reverse transcriptase was omitted from the RT reaction mixture; lane 4, M. tuberculosis DNA; and lane 5, no template. Lanes 1 and 6 contain 100-bp ladder DNA (GIBCO-BRL; Life Technologies Ltd, Paisley, United Kingdom).

Techniques Used: Expressing, Purification, Marker, Recombinant, Reverse Transcription Polymerase Chain Reaction

4) Product Images from "Isolation and In Vitro Differentiation of Conditionally Immortalized Murine Olfactory Receptor Neurons"

Article Title: Isolation and In Vitro Differentiation of Conditionally Immortalized Murine Olfactory Receptor Neurons

Journal: The Journal of Neuroscience

doi: 10.1523/JNEUROSCI.20-10-03695.2000

Amplification of olfactory-specific cDNAs with specific oligonucleotide primers. mRNA from cells grown in permissive ( 33 ) and nonpermissive ( 37 ) culture conditions and from tissue ( T ) was reverse transcribed (+) or incubated in the absence of enzyme (−). Specific oligonucleotide primers were then used to amplify the cDNA by PCR, and the products were run on 2% agarose gels beside a molecular marker ( M ). The primer pairs used were specific for β-tubulin, the olfactory cyclic nucleotide-gated channel subunits 1 and 2 ( OCNC1, OCNC2 ), the type III adenylate cyclase ( ACIII ), the olfactory-specific G-protein α subunit ( Golf ), a transcription factor in the olfactory epithelium ( OE1 ), and olfactory marker protein ( OMP ). For each of the primer pairs tested, no product was obtained from reverse transcriptase-free controls. For all other conditions, with the exception OMP amplification from cells in permissive conditions, specific products were obtained and confirmed by sequencing.
Figure Legend Snippet: Amplification of olfactory-specific cDNAs with specific oligonucleotide primers. mRNA from cells grown in permissive ( 33 ) and nonpermissive ( 37 ) culture conditions and from tissue ( T ) was reverse transcribed (+) or incubated in the absence of enzyme (−). Specific oligonucleotide primers were then used to amplify the cDNA by PCR, and the products were run on 2% agarose gels beside a molecular marker ( M ). The primer pairs used were specific for β-tubulin, the olfactory cyclic nucleotide-gated channel subunits 1 and 2 ( OCNC1, OCNC2 ), the type III adenylate cyclase ( ACIII ), the olfactory-specific G-protein α subunit ( Golf ), a transcription factor in the olfactory epithelium ( OE1 ), and olfactory marker protein ( OMP ). For each of the primer pairs tested, no product was obtained from reverse transcriptase-free controls. For all other conditions, with the exception OMP amplification from cells in permissive conditions, specific products were obtained and confirmed by sequencing.

Techniques Used: Amplification, Incubation, Polymerase Chain Reaction, Marker, Sequencing

5) Product Images from "Cloning, Sequence Analysis, and Characterization of the Genes Involved in Isoprimeverose Metabolism in Lactobacillus pentosus"

Article Title: Cloning, Sequence Analysis, and Characterization of the Genes Involved in Isoprimeverose Metabolism in Lactobacillus pentosus

Journal: Journal of Bacteriology

doi:

(A) Physical map and organization of the L. pentosus MD353 xylose regulon. The upper part shows the xylP xylQ cloning strategy. (B) Chromosomal situations of LPE1 and LPE2 integrants after integration of pLPA3 and pLPA4 plasmids. Their respective genotypes are indicated on the right (plus and minus signs indicate the presence and absence, respectively, of transcription of the genes). In both panels, arrows with right angles indicate the xylPQ and xylAB (xylose-inducible) promoters and the xylR (constitutive) promoters, and stem-loop structures indicate the putative transcriptional terminators. The sizes of the wild-type bacterial, LPE1, and LPE2 transcripts from the different promoters are given below each arrow (arrows with solid and dashed lines represent xylose-inducible and constitutive expression, respectively). The primers used for the inverse PCR, xylp5 and xylp3, are indicated by short open arrows below the Mun I fragment. bla and erm C, genes for ampicillin resistance and erythromycin resistance, respectively.
Figure Legend Snippet: (A) Physical map and organization of the L. pentosus MD353 xylose regulon. The upper part shows the xylP xylQ cloning strategy. (B) Chromosomal situations of LPE1 and LPE2 integrants after integration of pLPA3 and pLPA4 plasmids. Their respective genotypes are indicated on the right (plus and minus signs indicate the presence and absence, respectively, of transcription of the genes). In both panels, arrows with right angles indicate the xylPQ and xylAB (xylose-inducible) promoters and the xylR (constitutive) promoters, and stem-loop structures indicate the putative transcriptional terminators. The sizes of the wild-type bacterial, LPE1, and LPE2 transcripts from the different promoters are given below each arrow (arrows with solid and dashed lines represent xylose-inducible and constitutive expression, respectively). The primers used for the inverse PCR, xylp5 and xylp3, are indicated by short open arrows below the Mun I fragment. bla and erm C, genes for ampicillin resistance and erythromycin resistance, respectively.

Techniques Used: Clone Assay, Expressing, Inverse PCR

6) Product Images from "A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists"

Article Title: A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists

Journal: Analytical and Bioanalytical Chemistry

doi: 10.1007/s00216-007-1559-6

PCR controls. The PCR controls were performed as described in “PCR controls.” a Lanes 1, 6 and 11 contain a 100-bp ladder. PCR I was performed with primers on the backbone of the p406 plasmid and on the ARE 2 sequence. Lanes 2–5 are PCR I on the p406-ARE 2 - CYC1 -yEGFP reporter vector, the DNA that was isolated from the yeast transformant, the empty p406- CYC1 plasmid, and the DNA that was isolated from the empty yeast host (the nontransfected yeast cells), respectively. PCR II was performed with primers on the CYC1 promoter and the CYC1 terminator. Lanes 7–11 are PCR II on the reporter vector, the DNA from the yeast transformant, the empty p406- CYC1 plasmid, and the DNA from the empty yeast host respectively. b Lane 1 contains a 1-kb ladder. PCR III was performed with the primers on the human androgen receptor gene. Lanes 2–4 are PCR III on the p403-GPD-hAR expression vector, the DNA from the yeast transformant, and the DNA from the empty yeast host, respectively. yEGFP yeast enhanced green fluorescent protein
Figure Legend Snippet: PCR controls. The PCR controls were performed as described in “PCR controls.” a Lanes 1, 6 and 11 contain a 100-bp ladder. PCR I was performed with primers on the backbone of the p406 plasmid and on the ARE 2 sequence. Lanes 2–5 are PCR I on the p406-ARE 2 - CYC1 -yEGFP reporter vector, the DNA that was isolated from the yeast transformant, the empty p406- CYC1 plasmid, and the DNA that was isolated from the empty yeast host (the nontransfected yeast cells), respectively. PCR II was performed with primers on the CYC1 promoter and the CYC1 terminator. Lanes 7–11 are PCR II on the reporter vector, the DNA from the yeast transformant, the empty p406- CYC1 plasmid, and the DNA from the empty yeast host respectively. b Lane 1 contains a 1-kb ladder. PCR III was performed with the primers on the human androgen receptor gene. Lanes 2–4 are PCR III on the p403-GPD-hAR expression vector, the DNA from the yeast transformant, and the DNA from the empty yeast host, respectively. yEGFP yeast enhanced green fluorescent protein

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Isolation, Expressing

7) Product Images from "Intact EAV-HP Endogenous Retrovirus in Sonnerat's Jungle Fowl"

Article Title: Intact EAV-HP Endogenous Retrovirus in Sonnerat's Jungle Fowl

Journal: Journal of Virology

doi: 10.1128/JVI.75.4.2029-2032.2001

PCR amplification of 5-kbp EAV-HP provirus products with putative pol region sequences. (A) Schematic diagram showing the positions of oligonucleotide primers EVJFOR and 103ER used for PCR relative to a complete provirus. The sequence recognized by 103ER is deleted from the 4-kbp provirus types, indicated as chicken EAV-HP (ev/J), but is present in the type IV ev/J clone 4-1 sequence. Ψ, packaging signal; SD, splice donor; SA, splice acceptor. (B) Ethidium bromide-stained agarose gel of separated PCR products amplified from line 21 chicken, RJF, and SJF DNA. The 0.6-kbp product from RJF was amplified from the type IV provirus, and a 5-kbp product with putative pol sequences was amplified only from SJF DNA.
Figure Legend Snippet: PCR amplification of 5-kbp EAV-HP provirus products with putative pol region sequences. (A) Schematic diagram showing the positions of oligonucleotide primers EVJFOR and 103ER used for PCR relative to a complete provirus. The sequence recognized by 103ER is deleted from the 4-kbp provirus types, indicated as chicken EAV-HP (ev/J), but is present in the type IV ev/J clone 4-1 sequence. Ψ, packaging signal; SD, splice donor; SA, splice acceptor. (B) Ethidium bromide-stained agarose gel of separated PCR products amplified from line 21 chicken, RJF, and SJF DNA. The 0.6-kbp product from RJF was amplified from the type IV provirus, and a 5-kbp product with putative pol sequences was amplified only from SJF DNA.

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Staining, Agarose Gel Electrophoresis

PCR analysis of the gag-env deletion junctions of the EAV-HP proviruses from chickens and jungle fowl. (A) Diagram indicating the positions of primers in the gag region (H83REV) and in the env region (H8) flanking the deletion junctions of the 4-kbp EAV-HP provirus. (B) Ethidium bromide-stained PCR products amplified with the H83REV and H8 primers from two layer-type chicken lines, line 0 and brown leghorn (BRL), two meat-type chicken lines, 20 and 21, and two jungle fowl species, RJF and SJF. The EAV-HP1 clone and water were used as positive and negative controls, respectively. PCR products are indicated as I, II, and III, corresponding to the 4-kbp provirus types described in the text.
Figure Legend Snippet: PCR analysis of the gag-env deletion junctions of the EAV-HP proviruses from chickens and jungle fowl. (A) Diagram indicating the positions of primers in the gag region (H83REV) and in the env region (H8) flanking the deletion junctions of the 4-kbp EAV-HP provirus. (B) Ethidium bromide-stained PCR products amplified with the H83REV and H8 primers from two layer-type chicken lines, line 0 and brown leghorn (BRL), two meat-type chicken lines, 20 and 21, and two jungle fowl species, RJF and SJF. The EAV-HP1 clone and water were used as positive and negative controls, respectively. PCR products are indicated as I, II, and III, corresponding to the 4-kbp provirus types described in the text.

Techniques Used: Polymerase Chain Reaction, Staining, Amplification

8) Product Images from "Conversion of 3-Chlorocatechol by Various Catechol 2,3-Dioxygenases and Sequence Analysis of the Chlorocatechol Dioxygenase Region of Pseudomonas putida GJ31"

Article Title: Conversion of 3-Chlorocatechol by Various Catechol 2,3-Dioxygenases and Sequence Analysis of the Chlorocatechol Dioxygenase Region of Pseudomonas putida GJ31

Journal: Journal of Bacteriology

doi:

Schematic representation of the construction of hybrid catechol 2,3-dioxygenases (H1 to H10). The primers (P1 to P14) that were used in PCRs to generate DNA fragments that contained overlapping regions for the PCR fusions are indicated with arrows. □, polypeptide fragments derived from CbzE; , polypeptide fragments derived from TdnC. The amino acids of CbzE and TdnC between which the fusions were made are indicated.
Figure Legend Snippet: Schematic representation of the construction of hybrid catechol 2,3-dioxygenases (H1 to H10). The primers (P1 to P14) that were used in PCRs to generate DNA fragments that contained overlapping regions for the PCR fusions are indicated with arrows. □, polypeptide fragments derived from CbzE; , polypeptide fragments derived from TdnC. The amino acids of CbzE and TdnC between which the fusions were made are indicated.

Techniques Used: Polymerase Chain Reaction, Derivative Assay

9) Product Images from "Allele-Dependent Barley Grain ?-Amylase Activity 1"

Article Title: Allele-Dependent Barley Grain ?-Amylase Activity 1

Journal: Plant Physiology

doi:

Distribution of different β-amy1 alleles in domesticated barley. The β-amy1 allele-specific sequence of intron III was PCR amplified from genomic DNA of various barley cultivars. The fragments were size fractionated by acrylamide-gel electrophoresis. Lane M, Molecular size marker.
Figure Legend Snippet: Distribution of different β-amy1 alleles in domesticated barley. The β-amy1 allele-specific sequence of intron III was PCR amplified from genomic DNA of various barley cultivars. The fragments were size fractionated by acrylamide-gel electrophoresis. Lane M, Molecular size marker.

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Acrylamide Gel Assay, Electrophoresis, Marker

Identification of the β-amy1 allele in the offspring of the H. spontaneum × Adorra cross. Genomic DNA sequences from the parents cv Adorra (lane 1) and H. spontaneum PI 296897 (lane 2), and from the 15 individual lines #1 to #15 (lanes 3–17) were PCR amplified for β-amy1 intron III-specific sequences. The PCR products were size fractionated by acrylamide-gel electrophoresis. Lane M, Molecular size marker.
Figure Legend Snippet: Identification of the β-amy1 allele in the offspring of the H. spontaneum × Adorra cross. Genomic DNA sequences from the parents cv Adorra (lane 1) and H. spontaneum PI 296897 (lane 2), and from the 15 individual lines #1 to #15 (lanes 3–17) were PCR amplified for β-amy1 intron III-specific sequences. The PCR products were size fractionated by acrylamide-gel electrophoresis. Lane M, Molecular size marker.

Techniques Used: Polymerase Chain Reaction, Amplification, Acrylamide Gel Assay, Electrophoresis, Marker

Related Articles

Clone Assay:

Article Title: Intact EAV-HP Endogenous Retrovirus in Sonnerat's Jungle Fowl
Article Snippet: .. PCR was repeated using the Expand high-fidelity PCR system (Boehringer Mannheim) for cloning the 5-kbp SJF EAV-HP PCR product into the pGEM-T vector. .. Two cloned PCR products, designated EAV-JF1 and EAV-JF2, were isolated and sequenced.

Amplification:

Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression
Article Snippet: .. Strain NCM3467 (Δ ycjT ::Kanr ) which carries a deletion of 686 bp of the ycjT gene (,2267 bp total; deletion starts 544 bp downstream of the translational start codon) and an insertion of a kanamycin resistance cassette, used for selection, was obtained from the recD strain NCM3426 (see Table ) by the following steps. (i) The ycjT gene with flanking sequences of the ycjS and ycjU genes was amplified from strain MG1655 (CGSC 6300) with primers ycjT1 (5′-ACTAAGCTTAGATCCTGCCCAGGCGTACC) and ycjT2 (5′-AGTTCTAGAGCCAGAAGGCCGATAACCGC) and the Expand high-fidelity PCR system (Boehringer Mannheim-Roche). ..

Article Title: Cloning, Sequence Analysis, and Characterization of the Genes Involved in Isoprimeverose Metabolism in Lactobacillus pentosus
Article Snippet: .. One hundred nanograms of DNA and 20 pmol each of xylp5 (5′-GGCACCATATTTTTATGGAT-3′), complementary to codons 22 to 28 of xylP , and xylp3 (5′-GGAGTGAACGTTTCAGTTAT-3′), complementary to anticodons 30 to 35 of xylP , were used in the amplification reaction performed with the Expand high-fidelity PCR system (Boehringer Mannheim). .. The PCR fragment was sequenced by using the fmol DNA sequencing kit (Promega).

Generated:

Article Title: The R3 Region, One of Three Major Repetitive Regions of Human Herpesvirus 6, Is a Strong Enhancer of Immediate-Early Gene U95
Article Snippet: .. A 756-bp fragment and all deletion mutants of the U95 promoter were generated by PCR with the Expand high-fidelity PCR system (Boehringer Mannheim). .. Genomic DNA prepared from MT-4 cells infected with HHV-6B was used as the template.

Selection:

Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression
Article Snippet: .. Strain NCM3467 (Δ ycjT ::Kanr ) which carries a deletion of 686 bp of the ycjT gene (,2267 bp total; deletion starts 544 bp downstream of the translational start codon) and an insertion of a kanamycin resistance cassette, used for selection, was obtained from the recD strain NCM3426 (see Table ) by the following steps. (i) The ycjT gene with flanking sequences of the ycjS and ycjU genes was amplified from strain MG1655 (CGSC 6300) with primers ycjT1 (5′-ACTAAGCTTAGATCCTGCCCAGGCGTACC) and ycjT2 (5′-AGTTCTAGAGCCAGAAGGCCGATAACCGC) and the Expand high-fidelity PCR system (Boehringer Mannheim-Roche). ..

Polymerase Chain Reaction:

Article Title: A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists
Article Snippet: .. Full-length hAR cDNA was obtained using the Expand High Fidelity PCR system (Boehringer Mannheim) and an Eppendorf Mastercycler gradient. .. The sequence of the 5′-primer was 5′-GCTCTAGAATGGAAGTGCAGTTAGGGCTGGG-3′, containing a restriction site for Xba I just before the ATG start codon.

Article Title: Expression of a Gene for a Porin-Like Protein of the OmpA Family from Mycobacterium tuberculosis H37Rv
Article Snippet: .. PCR was performed with the Expand High Fidelity PCR system (Boehringer Mannheim Ltd., Lewes, E. Sussex, United Kingdom), using the 1.5 mM MgCl2 Expand buffer supplied with the kit. ..

Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression
Article Snippet: .. Strain NCM3467 (Δ ycjT ::Kanr ) which carries a deletion of 686 bp of the ycjT gene (,2267 bp total; deletion starts 544 bp downstream of the translational start codon) and an insertion of a kanamycin resistance cassette, used for selection, was obtained from the recD strain NCM3426 (see Table ) by the following steps. (i) The ycjT gene with flanking sequences of the ycjS and ycjU genes was amplified from strain MG1655 (CGSC 6300) with primers ycjT1 (5′-ACTAAGCTTAGATCCTGCCCAGGCGTACC) and ycjT2 (5′-AGTTCTAGAGCCAGAAGGCCGATAACCGC) and the Expand high-fidelity PCR system (Boehringer Mannheim-Roche). ..

Article Title: Intact EAV-HP Endogenous Retrovirus in Sonnerat's Jungle Fowl
Article Snippet: .. PCR was repeated using the Expand high-fidelity PCR system (Boehringer Mannheim) for cloning the 5-kbp SJF EAV-HP PCR product into the pGEM-T vector. .. Two cloned PCR products, designated EAV-JF1 and EAV-JF2, were isolated and sequenced.

Article Title: The R3 Region, One of Three Major Repetitive Regions of Human Herpesvirus 6, Is a Strong Enhancer of Immediate-Early Gene U95
Article Snippet: .. A 756-bp fragment and all deletion mutants of the U95 promoter were generated by PCR with the Expand high-fidelity PCR system (Boehringer Mannheim). .. Genomic DNA prepared from MT-4 cells infected with HHV-6B was used as the template.

Article Title: Cloning, Sequence Analysis, and Characterization of the Genes Involved in Isoprimeverose Metabolism in Lactobacillus pentosus
Article Snippet: .. One hundred nanograms of DNA and 20 pmol each of xylp5 (5′-GGCACCATATTTTTATGGAT-3′), complementary to codons 22 to 28 of xylP , and xylp3 (5′-GGAGTGAACGTTTCAGTTAT-3′), complementary to anticodons 30 to 35 of xylP , were used in the amplification reaction performed with the Expand high-fidelity PCR system (Boehringer Mannheim). .. The PCR fragment was sequenced by using the fmol DNA sequencing kit (Promega).

Article Title: Conversion of 3-Chlorocatechol by Various Catechol 2,3-Dioxygenases and Sequence Analysis of the Chlorocatechol Dioxygenase Region of Pseudomonas putida GJ31
Article Snippet: .. To ensure high fidelity of the PCR product, Pwo DNA polymerase or the Expand High Fidelity PCR system was used according to instructions of the manufacturer (Boehringer, Mannheim, Germany). ..

Plasmid Preparation:

Article Title: Intact EAV-HP Endogenous Retrovirus in Sonnerat's Jungle Fowl
Article Snippet: .. PCR was repeated using the Expand high-fidelity PCR system (Boehringer Mannheim) for cloning the 5-kbp SJF EAV-HP PCR product into the pGEM-T vector. .. Two cloned PCR products, designated EAV-JF1 and EAV-JF2, were isolated and sequenced.

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    Boehringer Mannheim expand high fidelity pcr system
    <t>PCR</t> amplification of fnr region from different <t>MG1655</t> isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.
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    PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

    Journal: Journal of Bacteriology

    Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression

    doi: 10.1128/JB.185.18.5611-5626.2003

    Figure Lengend Snippet: PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

    Article Snippet: Strain NCM3467 (Δ ycjT ::Kanr ) which carries a deletion of 686 bp of the ycjT gene (,2267 bp total; deletion starts 544 bp downstream of the translational start codon) and an insertion of a kanamycin resistance cassette, used for selection, was obtained from the recD strain NCM3426 (see Table ) by the following steps. (i) The ycjT gene with flanking sequences of the ycjS and ycjU genes was amplified from strain MG1655 (CGSC 6300) with primers ycjT1 (5′-ACTAAGCTTAGATCCTGCCCAGGCGTACC) and ycjT2 (5′-AGTTCTAGAGCCAGAAGGCCGATAACCGC) and the Expand high-fidelity PCR system (Boehringer Mannheim-Roche).

    Techniques: Polymerase Chain Reaction, Amplification

    Effect of repetition of the R3 unit on U95 promoter activity. (A) Schematic representation of PCR-generated R3 units containing NF-κB(R3) or NF-κB(TT) (termed R3-A and R3-B, respectively). (B) The left map represents the copy numbers of each R3 unit inserted immediately upstream of U95P-186. MT-4 cells were transfected with each construct and harvested 16 h later, and luciferase activities were measured. The right panel indicates the mean fold activities relative to that of pU95P-186-transfected cells in three independent experiments. The luciferase activities were normalized as described in Materials and Methods, and error bars plot the standard deviation for each set of triplicate samples.

    Journal: Journal of Virology

    Article Title: The R3 Region, One of Three Major Repetitive Regions of Human Herpesvirus 6, Is a Strong Enhancer of Immediate-Early Gene U95

    doi: 10.1128/JVI.75.21.10149-10160.2001

    Figure Lengend Snippet: Effect of repetition of the R3 unit on U95 promoter activity. (A) Schematic representation of PCR-generated R3 units containing NF-κB(R3) or NF-κB(TT) (termed R3-A and R3-B, respectively). (B) The left map represents the copy numbers of each R3 unit inserted immediately upstream of U95P-186. MT-4 cells were transfected with each construct and harvested 16 h later, and luciferase activities were measured. The right panel indicates the mean fold activities relative to that of pU95P-186-transfected cells in three independent experiments. The luciferase activities were normalized as described in Materials and Methods, and error bars plot the standard deviation for each set of triplicate samples.

    Article Snippet: A 756-bp fragment and all deletion mutants of the U95 promoter were generated by PCR with the Expand high-fidelity PCR system (Boehringer Mannheim).

    Techniques: Activity Assay, Polymerase Chain Reaction, Generated, Transfection, Construct, Luciferase, Standard Deviation

    RT-PCR analyses for the transcripts of U95, IE1, Pol, gH, and EF 1α in HST-infected CBMCs treated with CHX and PFA. Total RNAs prepared from mock-infected and HST-infected cells, which had been treated with CHX for 24 h or PFA for 24 h or left untreated, were used. The U95 band was detectable in the presence of CHX and PFA, as well as the IE1 band, while the bands of Pol and gH could not be detected in the presence of CHX. EF1α, the cellular endogenous gene, was transcribed under all conditions.

    Journal: Journal of Virology

    Article Title: The R3 Region, One of Three Major Repetitive Regions of Human Herpesvirus 6, Is a Strong Enhancer of Immediate-Early Gene U95

    doi: 10.1128/JVI.75.21.10149-10160.2001

    Figure Lengend Snippet: RT-PCR analyses for the transcripts of U95, IE1, Pol, gH, and EF 1α in HST-infected CBMCs treated with CHX and PFA. Total RNAs prepared from mock-infected and HST-infected cells, which had been treated with CHX for 24 h or PFA for 24 h or left untreated, were used. The U95 band was detectable in the presence of CHX and PFA, as well as the IE1 band, while the bands of Pol and gH could not be detected in the presence of CHX. EF1α, the cellular endogenous gene, was transcribed under all conditions.

    Article Snippet: A 756-bp fragment and all deletion mutants of the U95 promoter were generated by PCR with the Expand high-fidelity PCR system (Boehringer Mannheim).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Infection

    Expression of the gene for OmpATb in growing M. tuberculosis . Shown are gels scanned with a Leaf Lumina camera; images were processed on an Apple computer, using Adobe Photoshop version 3 and Macromedia Freehand version 5.5. (a) OmpATb and protein extracted from M. tuberculosis H37Rv were separated in SDS–10% polyacrylamide gels, blotted onto Immobilon-P membranes, and then detected with rabbit antiserum to truncated OmpATb. Lanes: 1, purified OmpATb; 2, protein from M. tuberculosis . The positions of marker proteins (not visible on the photograph) are indicated (in kilodaltons). Sample sizes were 50 ng for recombinant OmpATb and 100 μg for M. tuberculosis extract. (b) Gel of RT-PCR product obtained from mRNA of M. tuberculosis as described in Materials and Methods. Lanes 2 to 5 show products of PCRs with primers 1 and 3 and templates as follows: lane 2, reverse-transcribed M. tuberculosis RNA; lane 3, as for lane 2, but reverse transcriptase was omitted from the RT reaction mixture; lane 4, M. tuberculosis DNA; and lane 5, no template. Lanes 1 and 6 contain 100-bp ladder DNA (GIBCO-BRL; Life Technologies Ltd, Paisley, United Kingdom).

    Journal: Journal of Bacteriology

    Article Title: Expression of a Gene for a Porin-Like Protein of the OmpA Family from Mycobacterium tuberculosis H37Rv

    doi:

    Figure Lengend Snippet: Expression of the gene for OmpATb in growing M. tuberculosis . Shown are gels scanned with a Leaf Lumina camera; images were processed on an Apple computer, using Adobe Photoshop version 3 and Macromedia Freehand version 5.5. (a) OmpATb and protein extracted from M. tuberculosis H37Rv were separated in SDS–10% polyacrylamide gels, blotted onto Immobilon-P membranes, and then detected with rabbit antiserum to truncated OmpATb. Lanes: 1, purified OmpATb; 2, protein from M. tuberculosis . The positions of marker proteins (not visible on the photograph) are indicated (in kilodaltons). Sample sizes were 50 ng for recombinant OmpATb and 100 μg for M. tuberculosis extract. (b) Gel of RT-PCR product obtained from mRNA of M. tuberculosis as described in Materials and Methods. Lanes 2 to 5 show products of PCRs with primers 1 and 3 and templates as follows: lane 2, reverse-transcribed M. tuberculosis RNA; lane 3, as for lane 2, but reverse transcriptase was omitted from the RT reaction mixture; lane 4, M. tuberculosis DNA; and lane 5, no template. Lanes 1 and 6 contain 100-bp ladder DNA (GIBCO-BRL; Life Technologies Ltd, Paisley, United Kingdom).

    Article Snippet: PCR was performed with the Expand High Fidelity PCR system (Boehringer Mannheim Ltd., Lewes, E. Sussex, United Kingdom), using the 1.5 mM MgCl2 Expand buffer supplied with the kit.

    Techniques: Expressing, Purification, Marker, Recombinant, Reverse Transcription Polymerase Chain Reaction