Structured Review

Roche rapid dna ligation kit
MuPAR and <t>HuPAR2</t> chimeras reveal regions required for PERV-A 14/220* infection . MuPAR is not permissive for PERV-A binding and entry, while HuPAR2 is permissive for both. Chimeras were constructed by swapping regions of HuPAR2 (solid black) with the corresponding residues of MuPAR (hatched black). Constructs were tagged with either an N-terminal c-myc tag (open circle) or a C-terminal eGFP tag (gray oval), as a way to monitor expression. Chimeras were expressed in non-permissive SIRC cells and tested for PERV-A infection. Levels of infection were determined by PERV pol qPCR of 250 ng genomic <t>DNA</t> and compared to wild-type HuPAR2 and MuPAR. (-/+) indicates the status of PERV-A infection. The average PERV pol copy numbers and standard deviations ( n = 3) are shown for each. These chimeras revealed that the N-terminal 135 amino acids are critical for PERV-A 14/220* infection.
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Images

1) Product Images from "Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry"

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry

Journal: Retrovirology

doi: 10.1186/1742-4690-6-3

MuPAR and HuPAR2 chimeras reveal regions required for PERV-A 14/220* infection . MuPAR is not permissive for PERV-A binding and entry, while HuPAR2 is permissive for both. Chimeras were constructed by swapping regions of HuPAR2 (solid black) with the corresponding residues of MuPAR (hatched black). Constructs were tagged with either an N-terminal c-myc tag (open circle) or a C-terminal eGFP tag (gray oval), as a way to monitor expression. Chimeras were expressed in non-permissive SIRC cells and tested for PERV-A infection. Levels of infection were determined by PERV pol qPCR of 250 ng genomic DNA and compared to wild-type HuPAR2 and MuPAR. (-/+) indicates the status of PERV-A infection. The average PERV pol copy numbers and standard deviations ( n = 3) are shown for each. These chimeras revealed that the N-terminal 135 amino acids are critical for PERV-A 14/220* infection.
Figure Legend Snippet: MuPAR and HuPAR2 chimeras reveal regions required for PERV-A 14/220* infection . MuPAR is not permissive for PERV-A binding and entry, while HuPAR2 is permissive for both. Chimeras were constructed by swapping regions of HuPAR2 (solid black) with the corresponding residues of MuPAR (hatched black). Constructs were tagged with either an N-terminal c-myc tag (open circle) or a C-terminal eGFP tag (gray oval), as a way to monitor expression. Chimeras were expressed in non-permissive SIRC cells and tested for PERV-A infection. Levels of infection were determined by PERV pol qPCR of 250 ng genomic DNA and compared to wild-type HuPAR2 and MuPAR. (-/+) indicates the status of PERV-A infection. The average PERV pol copy numbers and standard deviations ( n = 3) are shown for each. These chimeras revealed that the N-terminal 135 amino acids are critical for PERV-A 14/220* infection.

Techniques Used: Infection, Binding Assay, Construct, Expressing, Real-time Polymerase Chain Reaction

HuPAR1 and HuPAR2 function for PERV-A 14/220* infection . HuPAR1 and HuPAR2 C-terminally tagged eGFP constructs were expressed in non-permissive SIRC cells. Stable lines were sorted by eGFP expression to yield cell populations with similar receptor expression levels. PERV pol copy number in 250 ng genomic DNA of infected SIRC/receptor-expressing cell lines was determined to assess receptor function. HuPAR2 PERV pol copy number was normalized by HuPAR1 PERV pol copy number in each experiment and expressed as percent of HuPAR1 function. The average function determined by three individual infection experiments with three replicates each is shown with standard errors. HuPAR2 is 11-fold more functional than HuPAR1 (p
Figure Legend Snippet: HuPAR1 and HuPAR2 function for PERV-A 14/220* infection . HuPAR1 and HuPAR2 C-terminally tagged eGFP constructs were expressed in non-permissive SIRC cells. Stable lines were sorted by eGFP expression to yield cell populations with similar receptor expression levels. PERV pol copy number in 250 ng genomic DNA of infected SIRC/receptor-expressing cell lines was determined to assess receptor function. HuPAR2 PERV pol copy number was normalized by HuPAR1 PERV pol copy number in each experiment and expressed as percent of HuPAR1 function. The average function determined by three individual infection experiments with three replicates each is shown with standard errors. HuPAR2 is 11-fold more functional than HuPAR1 (p

Techniques Used: Infection, Construct, Expressing, Functional Assay

2) Product Images from "Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection"

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx113

In vivo transposition in prokaryotic cells. ( A ) Scheme of the experimental method. The donor plasmid DNA, carrying a gene of interest (kanamycin resistance cassette, Kan R ) flanked with (IRs), contains a conditional origin of replication, oriR6K, which prevents replication in the recipient strain. Donor plasmid DNA and purified recombinant transposase (Mos1 or Mboumar-9) are co-transfected into bacterial cells, resulting in integration of the kanamycin cassette into the genomic DNA. ( B ) Kanamycin resistant colonies were obtained only if purified transposase was included in the transfection reaction. White colonies on dark background. ( C ) Relative efficiency of transposition observed after treatment of the reactions, prior to transfection, with proteinase K, phenol, no treatment and the control (no transposase added). Two technical repeats were performed for two biological repeats. ( D ) Agarose gel of the products of colony polymerase chain reaction (PCR) to detect the presence of the donor plasmid in the kanamycin resistant colonies. Clone 1 (Lane 4) has traces of the donor plasmid backbone detected. Positive control—a colony of Escherichia coli S17 λ pir carrying donor plasmid; negative control—a colony of the recipient strain E. coli DH10B. ( E ) Five analyzed clones are resistant to kanamycin, but sensitive to chloramphenicol—the plasmid backbone resistance. Controls as in (D). ( F ) Southern Blotting analysis of the digested genomic DNA from the kanamycin resistant clones hybridized with fluorescently labeled IR DNA. Negative control: genomic DNA of the recipient strain E. coli DH10B. ( G ) WebLogo alignment of 40 bp around the TA target nucleotides duplication of the 14 integration sites by Mos1 in the bacterial genome.
Figure Legend Snippet: In vivo transposition in prokaryotic cells. ( A ) Scheme of the experimental method. The donor plasmid DNA, carrying a gene of interest (kanamycin resistance cassette, Kan R ) flanked with (IRs), contains a conditional origin of replication, oriR6K, which prevents replication in the recipient strain. Donor plasmid DNA and purified recombinant transposase (Mos1 or Mboumar-9) are co-transfected into bacterial cells, resulting in integration of the kanamycin cassette into the genomic DNA. ( B ) Kanamycin resistant colonies were obtained only if purified transposase was included in the transfection reaction. White colonies on dark background. ( C ) Relative efficiency of transposition observed after treatment of the reactions, prior to transfection, with proteinase K, phenol, no treatment and the control (no transposase added). Two technical repeats were performed for two biological repeats. ( D ) Agarose gel of the products of colony polymerase chain reaction (PCR) to detect the presence of the donor plasmid in the kanamycin resistant colonies. Clone 1 (Lane 4) has traces of the donor plasmid backbone detected. Positive control—a colony of Escherichia coli S17 λ pir carrying donor plasmid; negative control—a colony of the recipient strain E. coli DH10B. ( E ) Five analyzed clones are resistant to kanamycin, but sensitive to chloramphenicol—the plasmid backbone resistance. Controls as in (D). ( F ) Southern Blotting analysis of the digested genomic DNA from the kanamycin resistant clones hybridized with fluorescently labeled IR DNA. Negative control: genomic DNA of the recipient strain E. coli DH10B. ( G ) WebLogo alignment of 40 bp around the TA target nucleotides duplication of the 14 integration sites by Mos1 in the bacterial genome.

Techniques Used: In Vivo, Plasmid Preparation, Purification, Recombinant, Transfection, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Positive Control, Negative Control, Clone Assay, Southern Blot, Labeling

3) Product Images from "Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load"

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.00742-14

Establishment of the cvPCR method and the limits of detection. (A) Authentic SFTSV strain HB29 viral RNA (HB29), the plasmid pCR-SFTSV-posicon (P.C.), which contains the artificial sequence, and the nontemplate control (H 2 O) were amplified by cvPCR using primer set 1 or 2. The PCR products were digested by EcoRI (+EcoRI). The sizes of the products were estimated by using a 2-log DNA ladder marker (NEB). (B and C) Isolated strains HB29, YG1, and SPL005, expanded in Vero cells, were diluted with serum from a healthy donor. The purified viral RNAs from the dilutions of virus-spiked serum were amplified. The resulting HB29, YG1, or SPL005 was amplified by primer set 1 (B) or 2 (C). The listed values are the dilutions of the viral strains. NTC, nontemplate control.
Figure Legend Snippet: Establishment of the cvPCR method and the limits of detection. (A) Authentic SFTSV strain HB29 viral RNA (HB29), the plasmid pCR-SFTSV-posicon (P.C.), which contains the artificial sequence, and the nontemplate control (H 2 O) were amplified by cvPCR using primer set 1 or 2. The PCR products were digested by EcoRI (+EcoRI). The sizes of the products were estimated by using a 2-log DNA ladder marker (NEB). (B and C) Isolated strains HB29, YG1, and SPL005, expanded in Vero cells, were diluted with serum from a healthy donor. The purified viral RNAs from the dilutions of virus-spiked serum were amplified. The resulting HB29, YG1, or SPL005 was amplified by primer set 1 (B) or 2 (C). The listed values are the dilutions of the viral strains. NTC, nontemplate control.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Sequencing, Amplification, Marker, Isolation, Purification

4) Product Images from "The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2"

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2

Journal: Glycobiology

doi: 10.1093/glycob/cwr110

Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The PCR-amplified DNA sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag
Figure Legend Snippet: Schematic representation of the expression constructs ( A ) pLgals1-hum-β3GN-T2 and ( B ) pLgals1-hum-MFng . The PCR-amplified DNA sequences were inserted in the pLgals1-Tev vector construct at the multicloning site, which is preceded by the 6HisTag

Techniques Used: Expressing, Construct, Polymerase Chain Reaction, Amplification, Plasmid Preparation

Related Articles

Clone Assay:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche). .. Human Hyal2 cDNA was amplified from pCMV6-HYAL2 (sc117754 OriGene) using the primers 5′-CCGGAATTC GCCACCATGCGGGCAGGCCCAGGCCCCACCG-3′ and 5′-ATAAGAATGCGGCCGC CTACAAGGTCCAGGTAAAGGCCAGGGC-3′ and cloned into pEGFP-N1-neo plasmid to replace EGFP fragment using EcoR1 and NotI restriction enzymes.

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: Paragraph title: Cloning of S. aureus azo1 gene and expression of Azo1 in E. coli ... The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche).

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol. .. Sequences were validated using the human GCNT2 cloning primers (Supplementary Table ).

Article Title: Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody
Article Snippet: .. The EPC1-cDNA product was cloned in the multicloning site (BamHI and EcoRI site) of pET28a plasmid using rapid DNA ligation kit (Roche, Germany). .. In order to express rEPC1, recombinant plasmid pET28a containing the EPC1 was transformed into competent E. coli BL21 cells.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: Plasmid DNA was isolated from the resistant clones and analyzed by restriction digest and Sanger sequencing. .. Two dilutions (1:10 and 1:20) were prepared for self-ligation of the genomic DNA fragments in a final volume of 20 μl at room temperature with Rapid DNA Ligation Kit (Roche) for 20 min. PCR was performed with Phusion High-Fidelity Polymerase (ThermoScientific) in a final volume of 20 μl, using 400 μM dNTPs (Invitrogen), 500 nM of primers (5΄-gtttcccgttgaatatggctc-3΄ and 5΄-actttctggctggatgatgg-3΄), 1 μl of DNA ligation mixture, 3% v/v Dimethyl sulfoxide (DMSO), 0.2 μl of Phusion Polymerase, dH2 O to 20 μl.

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Article Snippet: These PCR products were ligated by a Rapid DNA ligation kit (Roche Applied Science, Penzberg, Germany) after EcoRI digestion. .. The ligated PCR product was then further ligated into pCR-Blunt II-TOPO using a Zero Blunt TOPO PCR cloning kit (Life Technologies), according to the manufacturer's protocol.

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: Paragraph title: Cloning 3′UTR constructs and luciferase report assay ... The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions.

Article Title: The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-?B
Article Snippet: The resulting product was cloned into pCR2.1-TOPO (Invitrogen). .. The entire fragment spanning 3,539 base pairs was excised with the restriction enzyme PvuII (New England Biolabs) and ligated into the PmeI site of the targeting vector pOS (R. Thresher) with the Rapid DNA Ligation Kit (Roche).

Article Title: An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population
Article Snippet: Paragraph title: 4.1. cDNA Cloning and Plasmid Construction ... The digestion products of both the DNA of interest and the expression vector were ligated at room temperature for 5 min using the Rapid DNA Ligation Kit (Roche, Diagnostics GmbH, Mannheim, Germany).

Article Title: Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate
Article Snippet: DNA fragments used for cloning were amplified using Phusion high-fidelity DNA polymerase (NEB), according to the manufacturer's instructions. .. Unless otherwise stated, the antibiotic concentrations used were as follows: kanamycin (Km), 30 μg/ml; ampicillin (Amp), 100 μg/ml; tetracycline (Tc), 10 μg/ml; chloramphenicol (Cm), 10 μg/ml; streptomycin (Sm), 25 μg/ml; and trimethoprim (Tmp), 10 μg/ml. pRSF-oriT was generated by ligation (rapid DNA ligation kit; Roche) of amplified oriT and pRSFDuet-1 vector digested with the BssHII restriction enzyme. pUC-oriT was generated by ligation of amplified oriT and pUC18 vector digested with HindIII-HF and SacI-HF restriction enzymes.

Article Title: Restoration of anterior regeneration in a planarian with limited regenerative ability
Article Snippet: .. For cloning, 2–3 µL of PCR product was ligated with 70 ng of Eam1105I -digested pJC53.2 (Rapid DNA Ligation Kit, Roche, Mannheim, Germany) and used to transform DH5α. .. In vitro transcriptions with the appropriate RNA polymerase were performed using standard approaches with the addition of Digoxigenin-11-UTP (Roche, Mannheim, Germany).

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Amplification:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche). .. Human Hyal2 cDNA was amplified from pCMV6-HYAL2 (sc117754 OriGene) using the primers 5′-CCGGAATTC GCCACCATGCGGGCAGGCCCAGGCCCCACCG-3′ and 5′-ATAAGAATGCGGCCGC CTACAAGGTCCAGGTAAAGGCCAGGGC-3′ and cloned into pEGFP-N1-neo plasmid to replace EGFP fragment using EcoR1 and NotI restriction enzymes.

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: The amplification conditions were one cycle at 95 °C for 3 min, 30 cycles with each cycle including 30 s of melting at 95 °C, 30 s of annealing at 50 °C and 60 s of extension at 72 °C, and one final extension cycle at 72 °C for 5 min. .. The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche).

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: .. To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol. .. Sequences were validated using the human GCNT2 cloning primers (Supplementary Table ).

Article Title: Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody
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Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Article Snippet: Briefly, portions of two regions (positions 1044 to 1386 and 1267 to 1665) in the SFTSV YG1 small (S) segment were amplified with the addition of EcoRI site at each side (next to positions 1386 and 1267). .. These PCR products were ligated by a Rapid DNA ligation kit (Roche Applied Science, Penzberg, Germany) after EcoRI digestion.

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions. .. After E. coli transformation and construction amplification, plasmid DNA has been extracted by using the Plasmid Plus Maxi Kit (Qiagen, Italy), checked by PCR and by Sanger sequencing.

Article Title: An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population
Article Snippet: The amplified gene was cloned into pCR™2.1-TOPO-TA cloning vector (Invitrogen, Waltham, MA, USA) and transformed into TOP10 Escherichia coli cells. .. The digestion products of both the DNA of interest and the expression vector were ligated at room temperature for 5 min using the Rapid DNA Ligation Kit (Roche, Diagnostics GmbH, Mannheim, Germany).

Article Title: Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate
Article Snippet: .. Unless otherwise stated, the antibiotic concentrations used were as follows: kanamycin (Km), 30 μg/ml; ampicillin (Amp), 100 μg/ml; tetracycline (Tc), 10 μg/ml; chloramphenicol (Cm), 10 μg/ml; streptomycin (Sm), 25 μg/ml; and trimethoprim (Tmp), 10 μg/ml. pRSF-oriT was generated by ligation (rapid DNA ligation kit; Roche) of amplified oriT and pRSFDuet-1 vector digested with the BssHII restriction enzyme. pUC-oriT was generated by ligation of amplified oriT and pUC18 vector digested with HindIII-HF and SacI-HF restriction enzymes. .. Unless otherwise stated, all plasmids used in this study were generated by seamless cloning using the In-Fusion HD cloning kit (Clontech).

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry
Article Snippet: Vector and insert were ligated using the Rapid DNA Ligation Kit (Roche) to yield HuPAR1(HuPAR2 1–169)eGFP and HuPAR1(HuPAR2 170–448). .. HuPAR2 regions were introduced into the HuPAR1eGFP backbone by site-directed mutagenesis that required prior amplification of the HuPAR2 sequence to create a megaprimer with 5' and 3' homology to HuPAR1 nucleotide sequence based on [ ].

Article Title: Restoration of anterior regeneration in a planarian with limited regenerative ability
Article Snippet: Cloning To generate riboprobes, candidate genes were PCR amplified from cDNA generated from total RNA (iScript cDNA Synthesis Kit, Bio-Rad, Hercules, CA). .. For cloning, 2–3 µL of PCR product was ligated with 70 ng of Eam1105I -digested pJC53.2 (Rapid DNA Ligation Kit, Roche, Mannheim, Germany) and used to transform DH5α.

Positive Control:

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Article Snippet: To differentiate positive-control contamination of the samples during the cvPCR reaction, a pCR-SFTSV-posicon was constructed. .. These PCR products were ligated by a Rapid DNA ligation kit (Roche Applied Science, Penzberg, Germany) after EcoRI digestion.

Synthesized:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: The corresponding 63bp DNA oligonucleotides harboring the 19-mer hairpin sequence, loop sequence, polythymidine tract (U6 terminator), BamHI and HindIII restriction site overhangs were designed according to the user manual of pSilencer™ 2.1-U6 neo kit (Life Technologies) and chemically synthesized by IDT. .. The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche).

Article Title: Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody
Article Snippet: Then cDNA was synthesized using Reverse Transcriptase (Fermentas, Lithuania). .. The EPC1-cDNA product was cloned in the multicloning site (BamHI and EcoRI site) of pET28a plasmid using rapid DNA ligation kit (Roche, Germany).

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Construct:

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: To generate GCNT2 KD variants lentiviral shRNA constructs in the pLKO.1puro vector (#10878, Addgene) against GCNT2 ( ) or scrambled controls were purchased from the Mission collection (Sigma-Aldrich). .. To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol.

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Article Snippet: To differentiate positive-control contamination of the samples during the cvPCR reaction, a pCR-SFTSV-posicon was constructed. .. These PCR products were ligated by a Rapid DNA ligation kit (Roche Applied Science, Penzberg, Germany) after EcoRI digestion.

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: Paragraph title: Cloning 3′UTR constructs and luciferase report assay ... The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions.

Article Title: The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-?B
Article Snippet: The 3′ arm of homology of the Nlrc3 -deficient construct was obtained by PCR (Takara LA Taq) through the use of mouse 129/Sv genomic DNA with a forward primer for exon 4 (E4f; 5′-CCTGTGATGGAGTTGCTGGGCAGCGTGCTGAGTGG-3′) and a reverse primer for exon 6 (E6r; 5′-AGCCCCCGGTGGTCCAATGCTGTT ACTCCGGAGG-3′). .. The entire fragment spanning 3,539 base pairs was excised with the restriction enzyme PvuII (New England Biolabs) and ligated into the PmeI site of the targeting vector pOS (R. Thresher) with the Rapid DNA Ligation Kit (Roche).

Article Title: Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate
Article Snippet: Unless otherwise stated, the antibiotic concentrations used were as follows: kanamycin (Km), 30 μg/ml; ampicillin (Amp), 100 μg/ml; tetracycline (Tc), 10 μg/ml; chloramphenicol (Cm), 10 μg/ml; streptomycin (Sm), 25 μg/ml; and trimethoprim (Tmp), 10 μg/ml. pRSF-oriT was generated by ligation (rapid DNA ligation kit; Roche) of amplified oriT and pRSFDuet-1 vector digested with the BssHII restriction enzyme. pUC-oriT was generated by ligation of amplified oriT and pUC18 vector digested with HindIII-HF and SacI-HF restriction enzymes. .. In most cases, the constructs were generated by fusing two PCR fragments.

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry
Article Snippet: Paragraph title: Constructs ... Vector and insert were ligated using the Rapid DNA Ligation Kit (Roche) to yield HuPAR1(HuPAR2 1–169)eGFP and HuPAR1(HuPAR2 170–448).

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Real-time Polymerase Chain Reaction:

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Article Snippet: Positive-control plasmids for cvPCR and the RNA reference for qPCR that contained the target sequences of the cvPCR and qPCR primer-probe sets were prepared. .. These PCR products were ligated by a Rapid DNA ligation kit (Roche Applied Science, Penzberg, Germany) after EcoRI digestion.

Incubation:

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: For the annealing, 10 μM oligos have been diluted in annealing buffer (10 mM Tris-HCl, pH 7.5, 0,1 M NaCl, 1 mM EDTA), incubated at 96 °C for 5 minutes and gradually cooled down to room temperature. .. The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions.

Luciferase:

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: Paragraph title: Cloning 3′UTR constructs and luciferase report assay ... The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions.

Introduce:

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry
Article Snippet: Site-directed mutagenesis was used to introduce point mutations into the HuPAR2eGFP backbone and primer sequences are shown in Additional file , Table S2. .. Vector and insert were ligated using the Rapid DNA Ligation Kit (Roche) to yield HuPAR1(HuPAR2 1–169)eGFP and HuPAR1(HuPAR2 170–448).

Expressing:

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: Paragraph title: Cloning of S. aureus azo1 gene and expression of Azo1 in E. coli ... The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche).

Article Title: Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody
Article Snippet: PCR product, a band of 228 bp, was digested with BamHI and EcoRI and consequently the expression plasmid vector pET28a was double digested with the same restriction endonucleases as mentioned above. .. The EPC1-cDNA product was cloned in the multicloning site (BamHI and EcoRI site) of pET28a plasmid using rapid DNA ligation kit (Roche, Germany).

Article Title: An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population
Article Snippet: .. The digestion products of both the DNA of interest and the expression vector were ligated at room temperature for 5 min using the Rapid DNA Ligation Kit (Roche, Diagnostics GmbH, Mannheim, Germany). .. Ligated mixture was transformed into TOP10 E. coli cells selected with the same strategies as those described above.

Article Title: Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate
Article Snippet: The oriT and expression plasmids used in this study are described in Table S1 in the supplemental material. .. Unless otherwise stated, the antibiotic concentrations used were as follows: kanamycin (Km), 30 μg/ml; ampicillin (Amp), 100 μg/ml; tetracycline (Tc), 10 μg/ml; chloramphenicol (Cm), 10 μg/ml; streptomycin (Sm), 25 μg/ml; and trimethoprim (Tmp), 10 μg/ml. pRSF-oriT was generated by ligation (rapid DNA ligation kit; Roche) of amplified oriT and pRSFDuet-1 vector digested with the BssHII restriction enzyme. pUC-oriT was generated by ligation of amplified oriT and pUC18 vector digested with HindIII-HF and SacI-HF restriction enzymes.

Planar Chromatography:

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions. .. Lipofectamine 2000 has been employed for transfection of PLC/PRF/5 EveR and JHH6 EveR cells.

Transformation Assay:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche). .. Following transformation into Top10 competent cells (Life Technologies), successful ligation was confirmed using restriction digestion and DNA sequencing with M13F primer (5′-GTAAAACGACGGCCAGT-3′).

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche). .. E. coli NovaBlue (DE3) was transformed with the resulting plasmids.

Article Title: Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody
Article Snippet: The EPC1-cDNA product was cloned in the multicloning site (BamHI and EcoRI site) of pET28a plasmid using rapid DNA ligation kit (Roche, Germany). .. In order to express rEPC1, recombinant plasmid pET28a containing the EPC1 was transformed into competent E. coli BL21 cells.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: The whole reaction was transformed into E. coli DH10B chemically competent cells. .. Two dilutions (1:10 and 1:20) were prepared for self-ligation of the genomic DNA fragments in a final volume of 20 μl at room temperature with Rapid DNA Ligation Kit (Roche) for 20 min. PCR was performed with Phusion High-Fidelity Polymerase (ThermoScientific) in a final volume of 20 μl, using 400 μM dNTPs (Invitrogen), 500 nM of primers (5΄-gtttcccgttgaatatggctc-3΄ and 5΄-actttctggctggatgatgg-3΄), 1 μl of DNA ligation mixture, 3% v/v Dimethyl sulfoxide (DMSO), 0.2 μl of Phusion Polymerase, dH2 O to 20 μl.

Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Article Snippet: The appropriate restriction enzymes ( ) were used to digest both the vectors and inserts, and ligation was performed using the Rapid DNA Ligation Kit (Hoffman-La Roche Ltd., Basel, Switzerland). .. Ligated vectors were transformed into Subcloning Efficiency™ DH5α™ Competent Escherichia coli Cells (Life Technologies; Thermo Fisher Scientific, Burlington, ON, Canada), and E . coli cells that had been successfully transformed were selected for using the appropriate antibiotic ( ).

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions. .. After E. coli transformation and construction amplification, plasmid DNA has been extracted by using the Plasmid Plus Maxi Kit (Qiagen, Italy), checked by PCR and by Sanger sequencing.

Article Title: An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population
Article Snippet: Transformed cells were selected on LB agar plates containing 100 µg/mL ampicillin at 37 °C for 16–18 h. Positive transformants were inoculated into LB broth containing 100 µg/mL ampicillin for plasmid propagation. .. The digestion products of both the DNA of interest and the expression vector were ligated at room temperature for 5 min using the Rapid DNA Ligation Kit (Roche, Diagnostics GmbH, Mannheim, Germany).

Over Expression:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: Paragraph title: shRNA-mediated HAS knockdown and Hyal2 overexpression ... The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche).

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: For overexpression of Azo1 in E. coli , the PCR products were cleaved with Nco I and Bam HI (New England BioLabs). .. The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche).

Transfection:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche). .. Transfection grade plasmids were prepared with EndoFree plasmid maxi kit (Qiagen) and linearized with ScaI.

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: Viral supernatants were harvested 48–72 h after transfection and UACC62 melanoma cells were transduced in the presence of 6 µg/ml polybrene (#TR-1003-G, EMD Millipore) and selected with 1 µg/ml puromycin (#61-385-RA, Corning) to generate stable KD cell lines. .. To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol.

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions. .. Lipofectamine 2000 has been employed for transfection of PLC/PRF/5 EveR and JHH6 EveR cells.

Inverse PCR:

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: Inverse PCR: ∼1 μg of genomic DNA was digested with 1 μl of EcoRI (NEB) in 20 μl for 3 h at 37°C. .. Two dilutions (1:10 and 1:20) were prepared for self-ligation of the genomic DNA fragments in a final volume of 20 μl at room temperature with Rapid DNA Ligation Kit (Roche) for 20 min. PCR was performed with Phusion High-Fidelity Polymerase (ThermoScientific) in a final volume of 20 μl, using 400 μM dNTPs (Invitrogen), 500 nM of primers (5΄-gtttcccgttgaatatggctc-3΄ and 5΄-actttctggctggatgatgg-3΄), 1 μl of DNA ligation mixture, 3% v/v Dimethyl sulfoxide (DMSO), 0.2 μl of Phusion Polymerase, dH2 O to 20 μl.

DNA Ligation:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: .. The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche). .. Following transformation into Top10 competent cells (Life Technologies), successful ligation was confirmed using restriction digestion and DNA sequencing with M13F primer (5′-GTAAAACGACGGCCAGT-3′).

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: .. The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche). .. E. coli NovaBlue (DE3) was transformed with the resulting plasmids.

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: .. To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol. .. Sequences were validated using the human GCNT2 cloning primers (Supplementary Table ).

Article Title: Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody
Article Snippet: .. The EPC1-cDNA product was cloned in the multicloning site (BamHI and EcoRI site) of pET28a plasmid using rapid DNA ligation kit (Roche, Germany). .. In order to express rEPC1, recombinant plasmid pET28a containing the EPC1 was transformed into competent E. coli BL21 cells.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: .. Two dilutions (1:10 and 1:20) were prepared for self-ligation of the genomic DNA fragments in a final volume of 20 μl at room temperature with Rapid DNA Ligation Kit (Roche) for 20 min. PCR was performed with Phusion High-Fidelity Polymerase (ThermoScientific) in a final volume of 20 μl, using 400 μM dNTPs (Invitrogen), 500 nM of primers (5΄-gtttcccgttgaatatggctc-3΄ and 5΄-actttctggctggatgatgg-3΄), 1 μl of DNA ligation mixture, 3% v/v Dimethyl sulfoxide (DMSO), 0.2 μl of Phusion Polymerase, dH2 O to 20 μl. ..

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Article Snippet: .. These PCR products were ligated by a Rapid DNA ligation kit (Roche Applied Science, Penzberg, Germany) after EcoRI digestion. .. The ligated PCR product was then further ligated into pCR-Blunt II-TOPO using a Zero Blunt TOPO PCR cloning kit (Life Technologies), according to the manufacturer's protocol.

Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Article Snippet: .. The appropriate restriction enzymes ( ) were used to digest both the vectors and inserts, and ligation was performed using the Rapid DNA Ligation Kit (Hoffman-La Roche Ltd., Basel, Switzerland). .. Ligated vectors were transformed into Subcloning Efficiency™ DH5α™ Competent Escherichia coli Cells (Life Technologies; Thermo Fisher Scientific, Burlington, ON, Canada), and E . coli cells that had been successfully transformed were selected for using the appropriate antibiotic ( ).

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: .. The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions. .. After E. coli transformation and construction amplification, plasmid DNA has been extracted by using the Plasmid Plus Maxi Kit (Qiagen, Italy), checked by PCR and by Sanger sequencing.

Article Title: The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-?B
Article Snippet: .. The entire fragment spanning 3,539 base pairs was excised with the restriction enzyme PvuII (New England Biolabs) and ligated into the PmeI site of the targeting vector pOS (R. Thresher) with the Rapid DNA Ligation Kit (Roche). .. The 5′arm of homology was obtained by PCR with intron 1 forward primer 1 (I1f) (5′-AGAGCACTTGCTGTTCATGCTGAGGTCGCACGACTTG) and exon 2 reverse primer (E2r) (5′-TTCACCTGCTCAGCTGGGGAGCCAACACTGT-3′).

Article Title: An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population
Article Snippet: .. The digestion products of both the DNA of interest and the expression vector were ligated at room temperature for 5 min using the Rapid DNA Ligation Kit (Roche, Diagnostics GmbH, Mannheim, Germany). .. Ligated mixture was transformed into TOP10 E. coli cells selected with the same strategies as those described above.

Article Title: Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate
Article Snippet: .. Unless otherwise stated, the antibiotic concentrations used were as follows: kanamycin (Km), 30 μg/ml; ampicillin (Amp), 100 μg/ml; tetracycline (Tc), 10 μg/ml; chloramphenicol (Cm), 10 μg/ml; streptomycin (Sm), 25 μg/ml; and trimethoprim (Tmp), 10 μg/ml. pRSF-oriT was generated by ligation (rapid DNA ligation kit; Roche) of amplified oriT and pRSFDuet-1 vector digested with the BssHII restriction enzyme. pUC-oriT was generated by ligation of amplified oriT and pUC18 vector digested with HindIII-HF and SacI-HF restriction enzymes. .. Unless otherwise stated, all plasmids used in this study were generated by seamless cloning using the In-Fusion HD cloning kit (Clontech).

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry
Article Snippet: .. Vector and insert were ligated using the Rapid DNA Ligation Kit (Roche) to yield HuPAR1(HuPAR2 1–169)eGFP and HuPAR1(HuPAR2 170–448). .. HuPAR2 regions were introduced into the HuPAR1eGFP backbone by site-directed mutagenesis that required prior amplification of the HuPAR2 sequence to create a megaprimer with 5' and 3' homology to HuPAR1 nucleotide sequence based on [ ].

Article Title: Restoration of anterior regeneration in a planarian with limited regenerative ability
Article Snippet: .. For cloning, 2–3 µL of PCR product was ligated with 70 ng of Eam1105I -digested pJC53.2 (Rapid DNA Ligation Kit, Roche, Mannheim, Germany) and used to transform DH5α. .. In vitro transcriptions with the appropriate RNA polymerase were performed using standard approaches with the addition of Digoxigenin-11-UTP (Roche, Mannheim, Germany).

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Ligation:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche). .. Following transformation into Top10 competent cells (Life Technologies), successful ligation was confirmed using restriction digestion and DNA sequencing with M13F primer (5′-GTAAAACGACGGCCAGT-3′).

Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Article Snippet: .. The appropriate restriction enzymes ( ) were used to digest both the vectors and inserts, and ligation was performed using the Rapid DNA Ligation Kit (Hoffman-La Roche Ltd., Basel, Switzerland). .. Ligated vectors were transformed into Subcloning Efficiency™ DH5α™ Competent Escherichia coli Cells (Life Technologies; Thermo Fisher Scientific, Burlington, ON, Canada), and E . coli cells that had been successfully transformed were selected for using the appropriate antibiotic ( ).

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: .. The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions. .. After E. coli transformation and construction amplification, plasmid DNA has been extracted by using the Plasmid Plus Maxi Kit (Qiagen, Italy), checked by PCR and by Sanger sequencing.

Article Title: Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate
Article Snippet: .. Unless otherwise stated, the antibiotic concentrations used were as follows: kanamycin (Km), 30 μg/ml; ampicillin (Amp), 100 μg/ml; tetracycline (Tc), 10 μg/ml; chloramphenicol (Cm), 10 μg/ml; streptomycin (Sm), 25 μg/ml; and trimethoprim (Tmp), 10 μg/ml. pRSF-oriT was generated by ligation (rapid DNA ligation kit; Roche) of amplified oriT and pRSFDuet-1 vector digested with the BssHII restriction enzyme. pUC-oriT was generated by ligation of amplified oriT and pUC18 vector digested with HindIII-HF and SacI-HF restriction enzymes. .. Unless otherwise stated, all plasmids used in this study were generated by seamless cloning using the In-Fusion HD cloning kit (Clontech).

Infection:

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol. .. Viral supernatants were harvested 48–72 h after transfection and human A375 melanoma cells were infected with filtered retroviral supernatant and selected in 500 µg/ml Geneticin (#11811023, ThermoFisher).

Generated:

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: Lentiviral supernatants were generated by co-transfection of helper plasmids, pN8e-GagPolΔ8.1 and pNE8e-VSV/G, in the packaging cell line, HEK293-EBNA. .. To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol.

Article Title: Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate
Article Snippet: .. Unless otherwise stated, the antibiotic concentrations used were as follows: kanamycin (Km), 30 μg/ml; ampicillin (Amp), 100 μg/ml; tetracycline (Tc), 10 μg/ml; chloramphenicol (Cm), 10 μg/ml; streptomycin (Sm), 25 μg/ml; and trimethoprim (Tmp), 10 μg/ml. pRSF-oriT was generated by ligation (rapid DNA ligation kit; Roche) of amplified oriT and pRSFDuet-1 vector digested with the BssHII restriction enzyme. pUC-oriT was generated by ligation of amplified oriT and pUC18 vector digested with HindIII-HF and SacI-HF restriction enzymes. .. Unless otherwise stated, all plasmids used in this study were generated by seamless cloning using the In-Fusion HD cloning kit (Clontech).

Article Title: Restoration of anterior regeneration in a planarian with limited regenerative ability
Article Snippet: Cloning To generate riboprobes, candidate genes were PCR amplified from cDNA generated from total RNA (iScript cDNA Synthesis Kit, Bio-Rad, Hercules, CA). .. For cloning, 2–3 µL of PCR product was ligated with 70 ng of Eam1105I -digested pJC53.2 (Rapid DNA Ligation Kit, Roche, Mannheim, Germany) and used to transform DH5α.

DNA Sequencing:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche). .. Following transformation into Top10 competent cells (Life Technologies), successful ligation was confirmed using restriction digestion and DNA sequencing with M13F primer (5′-GTAAAACGACGGCCAGT-3′).

Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Article Snippet: The appropriate restriction enzymes ( ) were used to digest both the vectors and inserts, and ligation was performed using the Rapid DNA Ligation Kit (Hoffman-La Roche Ltd., Basel, Switzerland). .. Plasmids were isolated using the QIAprep Spin Miniprep Kit (Qiagen, Venlo, The Netherlands) and sequenced using Sanger DNA sequencing (Genome Quebec Innovation Centre, McGill University, Montreal, QC, Canada) to confirm that no mutations were introduced.

Article Title: An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population
Article Snippet: Plasmid was isolated and the presence of the DNA fragment of interest was determined by restriction enzyme digestion and DNA sequencing. .. The digestion products of both the DNA of interest and the expression vector were ligated at room temperature for 5 min using the Rapid DNA Ligation Kit (Roche, Diagnostics GmbH, Mannheim, Germany).

Polymerase Chain Reaction:

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: For overexpression of Azo1 in E. coli , the PCR products were cleaved with Nco I and Bam HI (New England BioLabs). .. The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche).

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: .. To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol. .. Sequences were validated using the human GCNT2 cloning primers (Supplementary Table ).

Article Title: Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody
Article Snippet: PCR product, a band of 228 bp, was digested with BamHI and EcoRI and consequently the expression plasmid vector pET28a was double digested with the same restriction endonucleases as mentioned above. .. The EPC1-cDNA product was cloned in the multicloning site (BamHI and EcoRI site) of pET28a plasmid using rapid DNA ligation kit (Roche, Germany).

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: .. Two dilutions (1:10 and 1:20) were prepared for self-ligation of the genomic DNA fragments in a final volume of 20 μl at room temperature with Rapid DNA Ligation Kit (Roche) for 20 min. PCR was performed with Phusion High-Fidelity Polymerase (ThermoScientific) in a final volume of 20 μl, using 400 μM dNTPs (Invitrogen), 500 nM of primers (5΄-gtttcccgttgaatatggctc-3΄ and 5΄-actttctggctggatgatgg-3΄), 1 μl of DNA ligation mixture, 3% v/v Dimethyl sulfoxide (DMSO), 0.2 μl of Phusion Polymerase, dH2 O to 20 μl. ..

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Article Snippet: .. These PCR products were ligated by a Rapid DNA ligation kit (Roche Applied Science, Penzberg, Germany) after EcoRI digestion. .. The ligated PCR product was then further ligated into pCR-Blunt II-TOPO using a Zero Blunt TOPO PCR cloning kit (Life Technologies), according to the manufacturer's protocol.

Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Article Snippet: Polymerase chain reaction (PCR) was performed to amplify the SCPX and SCP2 sequences and to create the appropriate restriction enzyme sites surrounding the desired sequence. .. The appropriate restriction enzymes ( ) were used to digest both the vectors and inserts, and ligation was performed using the Rapid DNA Ligation Kit (Hoffman-La Roche Ltd., Basel, Switzerland).

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions. .. After E. coli transformation and construction amplification, plasmid DNA has been extracted by using the Plasmid Plus Maxi Kit (Qiagen, Italy), checked by PCR and by Sanger sequencing.

Article Title: The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-?B
Article Snippet: The 3′ arm of homology of the Nlrc3 -deficient construct was obtained by PCR (Takara LA Taq) through the use of mouse 129/Sv genomic DNA with a forward primer for exon 4 (E4f; 5′-CCTGTGATGGAGTTGCTGGGCAGCGTGCTGAGTGG-3′) and a reverse primer for exon 6 (E6r; 5′-AGCCCCCGGTGGTCCAATGCTGTT ACTCCGGAGG-3′). .. The entire fragment spanning 3,539 base pairs was excised with the restriction enzyme PvuII (New England Biolabs) and ligated into the PmeI site of the targeting vector pOS (R. Thresher) with the Rapid DNA Ligation Kit (Roche).

Article Title: An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population
Article Snippet: The amplified gene was cloned into pCR™2.1-TOPO-TA cloning vector (Invitrogen, Waltham, MA, USA) and transformed into TOP10 Escherichia coli cells. .. The digestion products of both the DNA of interest and the expression vector were ligated at room temperature for 5 min using the Rapid DNA Ligation Kit (Roche, Diagnostics GmbH, Mannheim, Germany).

Article Title: Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate
Article Snippet: Unless otherwise stated, the antibiotic concentrations used were as follows: kanamycin (Km), 30 μg/ml; ampicillin (Amp), 100 μg/ml; tetracycline (Tc), 10 μg/ml; chloramphenicol (Cm), 10 μg/ml; streptomycin (Sm), 25 μg/ml; and trimethoprim (Tmp), 10 μg/ml. pRSF-oriT was generated by ligation (rapid DNA ligation kit; Roche) of amplified oriT and pRSFDuet-1 vector digested with the BssHII restriction enzyme. pUC-oriT was generated by ligation of amplified oriT and pUC18 vector digested with HindIII-HF and SacI-HF restriction enzymes. .. In most cases, the constructs were generated by fusing two PCR fragments.

Article Title: Restoration of anterior regeneration in a planarian with limited regenerative ability
Article Snippet: .. For cloning, 2–3 µL of PCR product was ligated with 70 ng of Eam1105I -digested pJC53.2 (Rapid DNA Ligation Kit, Roche, Mannheim, Germany) and used to transform DH5α. .. In vitro transcriptions with the appropriate RNA polymerase were performed using standard approaches with the addition of Digoxigenin-11-UTP (Roche, Mannheim, Germany).

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Recombinant:

Article Title: Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody
Article Snippet: Paragraph title: Recombinant protein EPC1 ... The EPC1-cDNA product was cloned in the multicloning site (BamHI and EcoRI site) of pET28a plasmid using rapid DNA ligation kit (Roche, Germany).

Imaging:

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. UDP-C2-keto-Gal and UDP-C2-keto-Glc were synthesized at the Chemical Biology Laboratory, Imaging Probe Development Center, National Institutes of Health, as described ( ).

In Vivo:

Article Title: The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-?B
Article Snippet: Paragraph title: Generation of Nlrc3 −/− mice and in vivo LPS treatment ... The entire fragment spanning 3,539 base pairs was excised with the restriction enzyme PvuII (New England Biolabs) and ligated into the PmeI site of the targeting vector pOS (R. Thresher) with the Rapid DNA Ligation Kit (Roche).

Mutagenesis:

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: Cloning 3′UTR constructs and luciferase report assay The plasmid pGL3-Control (Promega, Italy) encoding for the luciferase reporter gene under the control of a strong promoter, has been engineered to generate the pGL3-Luc-miR375bs-WT and pGL3-Luc-miR375bs-MUT vectors, harboring the wild type or the mutant c-MYC miR-375 seed sequence, respectively. .. The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions.

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry
Article Snippet: Site-directed mutagenesis was used to introduce point mutations into the HuPAR2eGFP backbone and primer sequences are shown in Additional file , Table S2. .. Vector and insert were ligated using the Rapid DNA Ligation Kit (Roche) to yield HuPAR1(HuPAR2 1–169)eGFP and HuPAR1(HuPAR2 170–448).

Isolation:

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche). .. The plasmids (pAZO1) were subsequently isolated and introduced into E. coli BL21-Gold(DE3)pLysS by transformation.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: Plasmid DNA was isolated from the resistant clones and analyzed by restriction digest and Sanger sequencing. .. Two dilutions (1:10 and 1:20) were prepared for self-ligation of the genomic DNA fragments in a final volume of 20 μl at room temperature with Rapid DNA Ligation Kit (Roche) for 20 min. PCR was performed with Phusion High-Fidelity Polymerase (ThermoScientific) in a final volume of 20 μl, using 400 μM dNTPs (Invitrogen), 500 nM of primers (5΄-gtttcccgttgaatatggctc-3΄ and 5΄-actttctggctggatgatgg-3΄), 1 μl of DNA ligation mixture, 3% v/v Dimethyl sulfoxide (DMSO), 0.2 μl of Phusion Polymerase, dH2 O to 20 μl.

Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Article Snippet: The appropriate restriction enzymes ( ) were used to digest both the vectors and inserts, and ligation was performed using the Rapid DNA Ligation Kit (Hoffman-La Roche Ltd., Basel, Switzerland). .. Plasmids were isolated using the QIAprep Spin Miniprep Kit (Qiagen, Venlo, The Netherlands) and sequenced using Sanger DNA sequencing (Genome Quebec Innovation Centre, McGill University, Montreal, QC, Canada) to confirm that no mutations were introduced.

Article Title: An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population
Article Snippet: Plasmid was isolated and the presence of the DNA fragment of interest was determined by restriction enzyme digestion and DNA sequencing. .. The digestion products of both the DNA of interest and the expression vector were ligated at room temperature for 5 min using the Rapid DNA Ligation Kit (Roche, Diagnostics GmbH, Mannheim, Germany).

Subcloning:

Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Article Snippet: The appropriate restriction enzymes ( ) were used to digest both the vectors and inserts, and ligation was performed using the Rapid DNA Ligation Kit (Hoffman-La Roche Ltd., Basel, Switzerland). .. Ligated vectors were transformed into Subcloning Efficiency™ DH5α™ Competent Escherichia coli Cells (Life Technologies; Thermo Fisher Scientific, Burlington, ON, Canada), and E . coli cells that had been successfully transformed were selected for using the appropriate antibiotic ( ).

Purification:

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: .. The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche). .. E. coli NovaBlue (DE3) was transformed with the resulting plasmids.

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry
Article Snippet: HuPAR1eGFP and HuPAR2eGFP were digested with Hind III/Xho I and Xho I/Kpn I. Fragments were excised from a 2% agarose gel and purified with the QIAquick Gel Extraction Kit (Qiagen). .. Vector and insert were ligated using the Rapid DNA Ligation Kit (Roche) to yield HuPAR1(HuPAR2 1–169)eGFP and HuPAR1(HuPAR2 170–448).

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Sequencing:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: The corresponding 63bp DNA oligonucleotides harboring the 19-mer hairpin sequence, loop sequence, polythymidine tract (U6 terminator), BamHI and HindIII restriction site overhangs were designed according to the user manual of pSilencer™ 2.1-U6 neo kit (Life Technologies) and chemically synthesized by IDT. .. The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche).

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche). .. DNA sequence analysis, translation and alignment with related genes and proteins were carried out using the Lasergene program (Version 5, dnastar ).

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: Paragraph title: Sequencing of prokaryotic genome integration sites ... Two dilutions (1:10 and 1:20) were prepared for self-ligation of the genomic DNA fragments in a final volume of 20 μl at room temperature with Rapid DNA Ligation Kit (Roche) for 20 min. PCR was performed with Phusion High-Fidelity Polymerase (ThermoScientific) in a final volume of 20 μl, using 400 μM dNTPs (Invitrogen), 500 nM of primers (5΄-gtttcccgttgaatatggctc-3΄ and 5΄-actttctggctggatgatgg-3΄), 1 μl of DNA ligation mixture, 3% v/v Dimethyl sulfoxide (DMSO), 0.2 μl of Phusion Polymerase, dH2 O to 20 μl.

Article Title: Sensitive and Specific PCR Systems for Detection of Both Chinese and Japanese Severe Fever with Thrombocytopenia Syndrome Virus Strains and Prediction of Patient Survival Based on Viral Load
Article Snippet: These PCR products were ligated by a Rapid DNA ligation kit (Roche Applied Science, Penzberg, Germany) after EcoRI digestion. .. The qPCR references for the primer-probe sets targeting the large (L), medium (M), or S segments were constructed with the insertion of the contamplicon sequence, which is, using a method described by Atkinson et al. , for the detection of RNA reference contamination by the contamplicon probe.

Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Article Snippet: Polymerase chain reaction (PCR) was performed to amplify the SCPX and SCP2 sequences and to create the appropriate restriction enzyme sites surrounding the desired sequence. .. The appropriate restriction enzymes ( ) were used to digest both the vectors and inserts, and ligation was performed using the Rapid DNA Ligation Kit (Hoffman-La Roche Ltd., Basel, Switzerland).

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: The c-MYC sequence of interest (5′UTR, Exone 1, Gene ID 4609) was: 5′ TGGAA GAGCCG GG CGA GCAGAGCT 3′, (in bold the miR-375 seed). pGL3-Control plasmid has been digested with XbaI enzyme. .. The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions.

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry
Article Snippet: Vector and insert were ligated using the Rapid DNA Ligation Kit (Roche) to yield HuPAR1(HuPAR2 1–169)eGFP and HuPAR1(HuPAR2 170–448). .. HuPAR2 regions were introduced into the HuPAR1eGFP backbone by site-directed mutagenesis that required prior amplification of the HuPAR2 sequence to create a megaprimer with 5' and 3' homology to HuPAR1 nucleotide sequence based on [ ].

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

shRNA:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: Paragraph title: shRNA-mediated HAS knockdown and Hyal2 overexpression ... The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche).

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: KD of human GCNT2 was achieved with the shRNA target sequences—shRNA #1: 5′-GCTAACAAGTTTGAGCTTAAT-3′ and shRNA #2: 5′-GCTCACCTCTATATTAGTTTA-3′. .. To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol.

Cotransfection:

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: Lentiviral supernatants were generated by co-transfection of helper plasmids, pN8e-GagPolΔ8.1 and pNE8e-VSV/G, in the packaging cell line, HEK293-EBNA. .. To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol.

IA:

Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Article Snippet: The pEGFP-N1 vector (Clontech Laboratories, Mountain View, CA) was used to create fluorescent fusion proteins of the putative mitochondrial targeting sequences of SCPX and SCP2 (pSCPX-mito-EGFP and pSCP2-mito-EGFP). lists the forward and reverse primers (Integrated DNA Technologies, Coralville, IA, USA), restriction enzymes (New England Biolabs, Ipswich, MA, USA), and antibiotics (Sigma-Aldrich Co., St Louis, MO, USA) used during the construction of each plasmid. .. The appropriate restriction enzymes ( ) were used to digest both the vectors and inserts, and ligation was performed using the Rapid DNA Ligation Kit (Hoffman-La Roche Ltd., Basel, Switzerland).

Mouse Assay:

Article Title: The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-?B
Article Snippet: Paragraph title: Generation of Nlrc3 −/− mice and in vivo LPS treatment ... The entire fragment spanning 3,539 base pairs was excised with the restriction enzyme PvuII (New England Biolabs) and ligated into the PmeI site of the targeting vector pOS (R. Thresher) with the Rapid DNA Ligation Kit (Roche).

Plasmid Preparation:

Article Title: High molecular weight hyaluronan mediates the cancer resistance of the naked mole-rat
Article Snippet: .. The complementary oligonucleotides were annealed and ligated to the pre-cut pSilencer2.1-U6 neo vector using the rapid DNA ligation kit (Roche). .. Following transformation into Top10 competent cells (Life Technologies), successful ligation was confirmed using restriction digestion and DNA sequencing with M13F primer (5′-GTAAAACGACGGCCAGT-3′).

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: Both strands of the DNA inserts were sequenced by walking from the ends of the inserts by using plasmid- and insert-specific primers. .. The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche).

Article Title: Loss of GCNT2/I-branched glycans enhances melanoma growth and survival
Article Snippet: .. To generate GCNT2 OE cell variants full length human GCNT2 cDNA (kindly provided by Tong Hao, Dana Farber Cancer Institute) was amplified using Platinum PCR SuperMix High Fidelity (#12532016, Life Technologies), digested and ligated into the retroviral plasmid, pLNCX2 (#631503, Clontech), using the Rapid DNA Ligation Kit (Roche) according to manufacturer’s protocol. .. Sequences were validated using the human GCNT2 cloning primers (Supplementary Table ).

Article Title: Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody
Article Snippet: .. The EPC1-cDNA product was cloned in the multicloning site (BamHI and EcoRI site) of pET28a plasmid using rapid DNA ligation kit (Roche, Germany). .. In order to express rEPC1, recombinant plasmid pET28a containing the EPC1 was transformed into competent E. coli BL21 cells.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: Plasmid DNA was isolated from the resistant clones and analyzed by restriction digest and Sanger sequencing. .. Two dilutions (1:10 and 1:20) were prepared for self-ligation of the genomic DNA fragments in a final volume of 20 μl at room temperature with Rapid DNA Ligation Kit (Roche) for 20 min. PCR was performed with Phusion High-Fidelity Polymerase (ThermoScientific) in a final volume of 20 μl, using 400 μM dNTPs (Invitrogen), 500 nM of primers (5΄-gtttcccgttgaatatggctc-3΄ and 5΄-actttctggctggatgatgg-3΄), 1 μl of DNA ligation mixture, 3% v/v Dimethyl sulfoxide (DMSO), 0.2 μl of Phusion Polymerase, dH2 O to 20 μl.

Article Title: Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells
Article Snippet: Paragraph title: Plasmid construction ... The appropriate restriction enzymes ( ) were used to digest both the vectors and inserts, and ligation was performed using the Rapid DNA Ligation Kit (Hoffman-La Roche Ltd., Basel, Switzerland).

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: The c-MYC sequence of interest (5′UTR, Exone 1, Gene ID 4609) was: 5′ TGGAA GAGCCG GG CGA GCAGAGCT 3′, (in bold the miR-375 seed). pGL3-Control plasmid has been digested with XbaI enzyme. .. The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions.

Article Title: The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-?B
Article Snippet: .. The entire fragment spanning 3,539 base pairs was excised with the restriction enzyme PvuII (New England Biolabs) and ligated into the PmeI site of the targeting vector pOS (R. Thresher) with the Rapid DNA Ligation Kit (Roche). .. The 5′arm of homology was obtained by PCR with intron 1 forward primer 1 (I1f) (5′-AGAGCACTTGCTGTTCATGCTGAGGTCGCACGACTTG) and exon 2 reverse primer (E2r) (5′-TTCACCTGCTCAGCTGGGGAGCCAACACTGT-3′).

Article Title: An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population
Article Snippet: .. The digestion products of both the DNA of interest and the expression vector were ligated at room temperature for 5 min using the Rapid DNA Ligation Kit (Roche, Diagnostics GmbH, Mannheim, Germany). .. Ligated mixture was transformed into TOP10 E. coli cells selected with the same strategies as those described above.

Article Title: Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate
Article Snippet: .. Unless otherwise stated, the antibiotic concentrations used were as follows: kanamycin (Km), 30 μg/ml; ampicillin (Amp), 100 μg/ml; tetracycline (Tc), 10 μg/ml; chloramphenicol (Cm), 10 μg/ml; streptomycin (Sm), 25 μg/ml; and trimethoprim (Tmp), 10 μg/ml. pRSF-oriT was generated by ligation (rapid DNA ligation kit; Roche) of amplified oriT and pRSFDuet-1 vector digested with the BssHII restriction enzyme. pUC-oriT was generated by ligation of amplified oriT and pUC18 vector digested with HindIII-HF and SacI-HF restriction enzymes. .. Unless otherwise stated, all plasmids used in this study were generated by seamless cloning using the In-Fusion HD cloning kit (Clontech).

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry
Article Snippet: .. Vector and insert were ligated using the Rapid DNA Ligation Kit (Roche) to yield HuPAR1(HuPAR2 1–169)eGFP and HuPAR1(HuPAR2 170–448). .. HuPAR2 regions were introduced into the HuPAR1eGFP backbone by site-directed mutagenesis that required prior amplification of the HuPAR2 sequence to create a megaprimer with 5' and 3' homology to HuPAR1 nucleotide sequence based on [ ].

Article Title: The N-acetyl-binding pocket of N-acetylglucosaminyltransferases also accommodates a sugar analog with a chemical handle at C2
Article Snippet: .. The DNA clones were obtained from Open Biosystems, Huntsville, Alabama and contained the coding sequence for hum-β3GN-T2 (accession number ) and hum-MFng (accession number ); the pLgals1 vector was constructed at our laboratory (25); Rosetta (DE3)pLysS cells were obtained from Novagen; XL2 blue ultra-competent cells from Stratagene; Taq-DNA polymerase, polymerase chain reaction (PCR) nucleotide mix and rapid DNA ligation kit from Roche Pharmaceuticals; DNA miniprep spin columns, PCR purification and low-melting agarose extraction kits from Qiagen; restriction enzymes from New England Biolabs, Inc.; Ampicillin, UDP-GlcNAc, UDP-GalNAc, free GlcNAc and pNP-α-Fuc from Sigma-Aldrich; UDP-[6-3 H]-GalNAc and UDP-[6-3 H]-GlcNAc from American Radiolabeled Chemicals; AG 1-X8 chloride resin 200–400 mesh from Bio-Rad; UDP-agarose gel from Calbiochem. .. DNA primers were synthesized by Integrated Technologies, Inc. Tev protease was obtained from Invitrogen.

Positron Emission Tomography:

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: .. The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche). .. E. coli NovaBlue (DE3) was transformed with the resulting plasmids.

Agarose Gel Electrophoresis:

Article Title: Biochemical and molecular characterization of an azoreductase from Staphylococcus aureus, a tetrameric NADPH-dependent flavoprotein
Article Snippet: .. The digested DNA was purified from the agarose gel and ligated into pET-11d with a rapid DNA ligation kit (Roche). .. E. coli NovaBlue (DE3) was transformed with the resulting plasmids.

Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
Article Snippet: Two dilutions (1:10 and 1:20) were prepared for self-ligation of the genomic DNA fragments in a final volume of 20 μl at room temperature with Rapid DNA Ligation Kit (Roche) for 20 min. PCR was performed with Phusion High-Fidelity Polymerase (ThermoScientific) in a final volume of 20 μl, using 400 μM dNTPs (Invitrogen), 500 nM of primers (5΄-gtttcccgttgaatatggctc-3΄ and 5΄-actttctggctggatgatgg-3΄), 1 μl of DNA ligation mixture, 3% v/v Dimethyl sulfoxide (DMSO), 0.2 μl of Phusion Polymerase, dH2 O to 20 μl. .. The whole PCR reaction was analyzed on a 1% (w/v) agarose gel.

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry
Article Snippet: HuPAR1eGFP and HuPAR2eGFP were digested with Hind III/Xho I and Xho I/Kpn I. Fragments were excised from a 2% agarose gel and purified with the QIAquick Gel Extraction Kit (Qiagen). .. Vector and insert were ligated using the Rapid DNA Ligation Kit (Roche) to yield HuPAR1(HuPAR2 1–169)eGFP and HuPAR1(HuPAR2 170–448).

In Vitro:

Article Title: Restoration of anterior regeneration in a planarian with limited regenerative ability
Article Snippet: For cloning, 2–3 µL of PCR product was ligated with 70 ng of Eam1105I -digested pJC53.2 (Rapid DNA Ligation Kit, Roche, Mannheim, Germany) and used to transform DH5α. .. In vitro transcriptions with the appropriate RNA polymerase were performed using standard approaches with the addition of Digoxigenin-11-UTP (Roche, Mannheim, Germany).

Spectrophotometry:

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions. .. The construct was further amplified, extracted with the GenElute HP Plamid Maxiprep Kit (Sigma Aldrich, Italy) and quantified by using NanoDrop spectrophotometer.

Concentration Assay:

Article Title: Evaluation of Dot Immunogold Filtration Assay (DIGFA) By Recombinant Protein EPC1 for Anti- Echinococcus granulosus IgG Antibody
Article Snippet: The EPC1-cDNA product was cloned in the multicloning site (BamHI and EcoRI site) of pET28a plasmid using rapid DNA ligation kit (Roche, Germany). .. After that, Protein expression was induced at 33°C for 6 h in the presence of isopropyl-b-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM.

Article Title: Vitamin D reverts resistance to the mTOR inhibitor everolimus in hepatocellular carcinoma through the activation of a miR-375/oncogenes circuit
Article Snippet: Sense and antisense nucleotides were resuspended in H2 O to a final concentration of 100 μM. .. The ligation reactions were performed with the Rapid DNA ligation kit (Roche, Italy) according to manufacturers’ instructions.

Gel Extraction:

Article Title: Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry
Article Snippet: HuPAR1eGFP and HuPAR2eGFP were digested with Hind III/Xho I and Xho I/Kpn I. Fragments were excised from a 2% agarose gel and purified with the QIAquick Gel Extraction Kit (Qiagen). .. Vector and insert were ligated using the Rapid DNA Ligation Kit (Roche) to yield HuPAR1(HuPAR2 1–169)eGFP and HuPAR1(HuPAR2 170–448).

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    Roche rapid dna ligation kit
    <t>NFI-A</t> binds to the L1 gene in vivo . A , ChIP analysis of N2A cells expressing Myc-NFI-A. L1 , <t>NFI-A-DNA</t> complexes in the region of the L1 gene containing the NFI full binding site were assayed by PCR using primers indicated in Fig. 1A. Lanes: NFI-A bs , precipitation from Myc-NFI-A bs-transfected cells; NFI-A st , precipitation from Myc-NFI-A st-transfected cells; mock , precipitation from mock-transfected cells; pos , positive control (mouse genomic DNA as PCR template); water , water control (without PCR template). Chst8 and Chst11 , To further confirm the specificity of ChIP analysis, ChIP samples were analyzed by PCR using primers amplifying parts of the mouse Chst8 and Chst11 genes, respectively. Note the absence of PCR signals for Chst8 and Chst11 in the ChIP sample lanes. B , Expression of Myc-tagged NFI-A isoforms in N2A cells. N2A cells were transfected with 4.8 μg of NFI-A cDNA expression vectors to express Myc-NFI-A st or Myc-NFI-A bs, respectively, or with 4.8 μg pCMX plasmid (
    Rapid Dna Ligation Kit, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    NFI-A binds to the L1 gene in vivo . A , ChIP analysis of N2A cells expressing Myc-NFI-A. L1 , NFI-A-DNA complexes in the region of the L1 gene containing the NFI full binding site were assayed by PCR using primers indicated in Fig. 1A. Lanes: NFI-A bs , precipitation from Myc-NFI-A bs-transfected cells; NFI-A st , precipitation from Myc-NFI-A st-transfected cells; mock , precipitation from mock-transfected cells; pos , positive control (mouse genomic DNA as PCR template); water , water control (without PCR template). Chst8 and Chst11 , To further confirm the specificity of ChIP analysis, ChIP samples were analyzed by PCR using primers amplifying parts of the mouse Chst8 and Chst11 genes, respectively. Note the absence of PCR signals for Chst8 and Chst11 in the ChIP sample lanes. B , Expression of Myc-tagged NFI-A isoforms in N2A cells. N2A cells were transfected with 4.8 μg of NFI-A cDNA expression vectors to express Myc-NFI-A st or Myc-NFI-A bs, respectively, or with 4.8 μg pCMX plasmid (

    Journal: BMC Molecular Biology

    Article Title: Nuclear factor I-A represses expression of the cell adhesion molecule L1

    doi: 10.1186/1471-2199-10-107

    Figure Lengend Snippet: NFI-A binds to the L1 gene in vivo . A , ChIP analysis of N2A cells expressing Myc-NFI-A. L1 , NFI-A-DNA complexes in the region of the L1 gene containing the NFI full binding site were assayed by PCR using primers indicated in Fig. 1A. Lanes: NFI-A bs , precipitation from Myc-NFI-A bs-transfected cells; NFI-A st , precipitation from Myc-NFI-A st-transfected cells; mock , precipitation from mock-transfected cells; pos , positive control (mouse genomic DNA as PCR template); water , water control (without PCR template). Chst8 and Chst11 , To further confirm the specificity of ChIP analysis, ChIP samples were analyzed by PCR using primers amplifying parts of the mouse Chst8 and Chst11 genes, respectively. Note the absence of PCR signals for Chst8 and Chst11 in the ChIP sample lanes. B , Expression of Myc-tagged NFI-A isoforms in N2A cells. N2A cells were transfected with 4.8 μg of NFI-A cDNA expression vectors to express Myc-NFI-A st or Myc-NFI-A bs, respectively, or with 4.8 μg pCMX plasmid ("mock"). 48 h after transfection, cell were lysed and whole cell extracts were analyzed on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and probed with anti-Myc antibody for detection of recombinant Myc-NFI-A and with anti-GAPDH antibody to check for loading of comparable protein amounts.

    Article Snippet: The resulting double-stranded oligonucleotide was added to a 5-10 fold excess of the respective Not I/Sfi I-digested NFI-A expression vector, and ligated using the Rapid DNA Ligation Kit.

    Techniques: In Vivo, Chromatin Immunoprecipitation, Expressing, Binding Assay, Polymerase Chain Reaction, Transfection, Positive Control, Plasmid Preparation, SDS Page, Recombinant

    NFI-A binds to its full binding site in the mouse L1 gene in vitro . 32 P-labeled oligonucleotides used for EMSA: L: full NFI binding site in mouse L1 gene with flanking sequences; Lc: point-mutated variant of L; N: idealized NFI binding site with flanking sequences [ 18 ]; Nc: point-mutated variant of N; C: oligonucleotide without NFI recognition motif (SIS oligonucleotide). NFI binding site sequences are capitalized , point mutations are shown in boldface . The sequence of one strand is shown after the fill-in reaction. Oligonucleotides were incubated with extracts from CHO cells expressing NFI-A bs or NFI-A st, or from mock-transfected cells (CMV). Only the upper and lower parts of the autoradiogram are shown, the middle section, which did not exhibit any signals, was cut out for reasons of space. Arrows A: HA-NFI-A-DNA complexes; arrow B: complexes of endogenous NFI and DNA; arrows C and D: free oligonucleotide DNA. Note that that the free SIS oligonucleotide migrates slightly slower ( arrow C) than the other free oligonucleotides ( arrow D) due to its higher molecular weight.

    Journal: BMC Molecular Biology

    Article Title: Nuclear factor I-A represses expression of the cell adhesion molecule L1

    doi: 10.1186/1471-2199-10-107

    Figure Lengend Snippet: NFI-A binds to its full binding site in the mouse L1 gene in vitro . 32 P-labeled oligonucleotides used for EMSA: L: full NFI binding site in mouse L1 gene with flanking sequences; Lc: point-mutated variant of L; N: idealized NFI binding site with flanking sequences [ 18 ]; Nc: point-mutated variant of N; C: oligonucleotide without NFI recognition motif (SIS oligonucleotide). NFI binding site sequences are capitalized , point mutations are shown in boldface . The sequence of one strand is shown after the fill-in reaction. Oligonucleotides were incubated with extracts from CHO cells expressing NFI-A bs or NFI-A st, or from mock-transfected cells (CMV). Only the upper and lower parts of the autoradiogram are shown, the middle section, which did not exhibit any signals, was cut out for reasons of space. Arrows A: HA-NFI-A-DNA complexes; arrow B: complexes of endogenous NFI and DNA; arrows C and D: free oligonucleotide DNA. Note that that the free SIS oligonucleotide migrates slightly slower ( arrow C) than the other free oligonucleotides ( arrow D) due to its higher molecular weight.

    Article Snippet: The resulting double-stranded oligonucleotide was added to a 5-10 fold excess of the respective Not I/Sfi I-digested NFI-A expression vector, and ligated using the Rapid DNA Ligation Kit.

    Techniques: Binding Assay, In Vitro, Labeling, Variant Assay, Sequencing, Incubation, Expressing, Transfection, Molecular Weight

    In vitro binding of NFI-A to its full binding site in the mouse L1 gene is confirmed by supershift and competition assays . EMSAs were performed with 32 P-labeled oligonucleotides indicated above the autoradiograms (for a description, see Fig. 3). In both images ( A-B ), only the upper and lower parts of the respective autoradiogram are shown, the middle sections, which did not exhibit any signals, were cut out for reasons of space. A , Supershift experiment. NFI-A st

    Journal: BMC Molecular Biology

    Article Title: Nuclear factor I-A represses expression of the cell adhesion molecule L1

    doi: 10.1186/1471-2199-10-107

    Figure Lengend Snippet: In vitro binding of NFI-A to its full binding site in the mouse L1 gene is confirmed by supershift and competition assays . EMSAs were performed with 32 P-labeled oligonucleotides indicated above the autoradiograms (for a description, see Fig. 3). In both images ( A-B ), only the upper and lower parts of the respective autoradiogram are shown, the middle sections, which did not exhibit any signals, were cut out for reasons of space. A , Supershift experiment. NFI-A st "+": oligonucleotides were incubated with extracts from CHO cells transfected with pCHNFI-A (NFI-A st); NFI-A st "-": oligonucleotides were not pre-incubated with cell extracts (negative control). Where indicated, anti-HA antibody was added. Arrow a points at anti-HA-antibody/NFI-A/DNA super-shifted complexes, while arrow b indicates NFI-A/DNA complexes. B , Competition analysis with the radioactively labeled NFI binding site from the mouse L1 gene regulatory region (L) and various unlabeled competitor oligonucleotides (N, Nc, L, Lc; see Fig. 3 for a description). The 32 P-labeled L oligonucleotide was incubated with extracts from CHO cells transfected with pCHNFI-A (NFI-A st) in the presence of different molar excesses of the unlabeled competitor oligonucleotides as indicated above the autoradiogram. A "-" indicates that oligonucleotides were not pre-incubated with cell extracts (negative control). The arrow points at the main band representing NFI-A-DNA complexes.

    Article Snippet: The resulting double-stranded oligonucleotide was added to a 5-10 fold excess of the respective Not I/Sfi I-digested NFI-A expression vector, and ligated using the Rapid DNA Ligation Kit.

    Techniques: In Vitro, Binding Assay, Labeling, Incubation, Transfection, Negative Control

    NFI-A represses mouse L1 gene expression . Results of luciferase-based reporter gene assays in N2A cells transfected with L1-11. The L1 reporter plasmid L1-11 contains 2943 bp upstream of exon 1 of the mouse L1 gene, exon 1, intron 1, exon 2 with the luciferase cDNA inserted to replace the L1 start codon by the luciferase start codon, intron 2 including the neural restrictive silencer element, exon 3, intron 3 and exon 4. beta Gal: coexpression of beta-galactosidase (negative control) ( n = 98); NFI-A bs: coexpression of NFI-A bs ( n = 91); NFI-A bs del: coexpression of NFI-A bs del ( n = 35). For titration of beta Gal with NFI-A bs, the beta Gal expression plasmid was gradually replaced with the NFI-A bs expression plasmid at the ratios indicated below, leaving the total amount of transfected DNA constant.

    Journal: BMC Molecular Biology

    Article Title: Nuclear factor I-A represses expression of the cell adhesion molecule L1

    doi: 10.1186/1471-2199-10-107

    Figure Lengend Snippet: NFI-A represses mouse L1 gene expression . Results of luciferase-based reporter gene assays in N2A cells transfected with L1-11. The L1 reporter plasmid L1-11 contains 2943 bp upstream of exon 1 of the mouse L1 gene, exon 1, intron 1, exon 2 with the luciferase cDNA inserted to replace the L1 start codon by the luciferase start codon, intron 2 including the neural restrictive silencer element, exon 3, intron 3 and exon 4. beta Gal: coexpression of beta-galactosidase (negative control) ( n = 98); NFI-A bs: coexpression of NFI-A bs ( n = 91); NFI-A bs del: coexpression of NFI-A bs del ( n = 35). For titration of beta Gal with NFI-A bs, the beta Gal expression plasmid was gradually replaced with the NFI-A bs expression plasmid at the ratios indicated below, leaving the total amount of transfected DNA constant. "1", bs:Gal = 1:59 ( n = 28); "2", bs:Gal = 1:11 ( n = 35); "3", bs:Gal = 1:5 ( n = 49); "4", bs:Gal = 1:2 ( n = 21); "5", bs:Gal = 1:1 ( n = 49); "6", bs:Gal = 2:1 ( n = 21). Data were acquired from 3 (bs:Gal = 1:2) to 9 (beta Gal, NFI-A bs) independent experiments. For normalization, luciferase activity was divided by the fluorescence of coexpressed EGFP. The relative luciferase activity of the beta Gal-transfected samples was set to 1. **, p

    Article Snippet: The resulting double-stranded oligonucleotide was added to a 5-10 fold excess of the respective Not I/Sfi I-digested NFI-A expression vector, and ligated using the Rapid DNA Ligation Kit.

    Techniques: Expressing, Luciferase, Transfection, Plasmid Preparation, Negative Control, Titration, Activity Assay, Fluorescence