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Activities of the wild-type and brachymorphic SK2 enzyme. ( A ) ATP sulfurylase activity as measured by the reverse sulfurylase assay where formation of ATP is monitored. ( B ) APS kinase activity is measured as the amount of PAPS generated from APS and ATP. ( C ) PAPS synthetase activity is measured as the amount of PAPS generated starting from free sulfate and ATP. Controls in A and B represent DE3 cells and DE3 cells with <t>pET-15b</t> vector.
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Images

1) Product Images from "A member of a family of sulfate-activating enzymes causes murine brachymorphism"

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Activities of the wild-type and brachymorphic SK2 enzyme. ( A ) ATP sulfurylase activity as measured by the reverse sulfurylase assay where formation of ATP is monitored. ( B ) APS kinase activity is measured as the amount of PAPS generated from APS and ATP. ( C ) PAPS synthetase activity is measured as the amount of PAPS generated starting from free sulfate and ATP. Controls in A and B represent DE3 cells and DE3 cells with pET-15b vector.
Figure Legend Snippet: Activities of the wild-type and brachymorphic SK2 enzyme. ( A ) ATP sulfurylase activity as measured by the reverse sulfurylase assay where formation of ATP is monitored. ( B ) APS kinase activity is measured as the amount of PAPS generated from APS and ATP. ( C ) PAPS synthetase activity is measured as the amount of PAPS generated starting from free sulfate and ATP. Controls in A and B represent DE3 cells and DE3 cells with pET-15b vector.

Techniques Used: Activity Assay, Papanicolaou Stain, Generated, Positron Emission Tomography, Plasmid Preparation

2) Product Images from "A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis"

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-03-0138

Ct-RBD-1 interacts with pre-rRNA. (A) Schematic representation of the C. tentans rRNA gene. The regions of the pre-rRNA used for binding studies with Ct-RBD-1 are shown below the gene and numbered 1–6. 32 P-Labeled RNAs were obtained by in vitro transcription of PCR fragments representing the different regions. The lengths of the RNAs were 438, 442, 477, 412, 519, and 536 nucleotides for RNA 1 to RNA 6, respectively. (B) Filter binding assay with Ct-RBD-1 and RNAs representing different pre-rRNA regions and control RNAs. Then 2–3 fmol of each RNA (in molecules) was incubated with increasing amounts of Ct-RBD-1 and filtered through nitrocellulose filters. The percentage retained RNA was plotted against the concentration of protein. Control RNAs were obtained by transcribing restriction enzyme cleaved pET-15b plasmid DNA (control 2, 430 nucleotides) or a PCR fragment representing a globin gene construct (control 1, 353 nucleotides). A third control RNA (transcribed from the plasmid pBluescript) behaved similarly to control RNA 2 (our unpublished data). The values shown are the averages of two separate measurements. Binding of RNA containing part of the 5′ ETS or ITS 2 (RNA 1 and RNA 5) bound Ct-RBD-1 with similar high affinities ( K d value of ∼5 nM). RNA representing the ITS 1 (RNA 4) bound with slightly less high affinity ( K d value of 10–15 nM). Binding of RNA containing 18S (RNA 2 and 3) or 28S (RNA 6) rRNA sequences was not different compared with the control RNAs.
Figure Legend Snippet: Ct-RBD-1 interacts with pre-rRNA. (A) Schematic representation of the C. tentans rRNA gene. The regions of the pre-rRNA used for binding studies with Ct-RBD-1 are shown below the gene and numbered 1–6. 32 P-Labeled RNAs were obtained by in vitro transcription of PCR fragments representing the different regions. The lengths of the RNAs were 438, 442, 477, 412, 519, and 536 nucleotides for RNA 1 to RNA 6, respectively. (B) Filter binding assay with Ct-RBD-1 and RNAs representing different pre-rRNA regions and control RNAs. Then 2–3 fmol of each RNA (in molecules) was incubated with increasing amounts of Ct-RBD-1 and filtered through nitrocellulose filters. The percentage retained RNA was plotted against the concentration of protein. Control RNAs were obtained by transcribing restriction enzyme cleaved pET-15b plasmid DNA (control 2, 430 nucleotides) or a PCR fragment representing a globin gene construct (control 1, 353 nucleotides). A third control RNA (transcribed from the plasmid pBluescript) behaved similarly to control RNA 2 (our unpublished data). The values shown are the averages of two separate measurements. Binding of RNA containing part of the 5′ ETS or ITS 2 (RNA 1 and RNA 5) bound Ct-RBD-1 with similar high affinities ( K d value of ∼5 nM). RNA representing the ITS 1 (RNA 4) bound with slightly less high affinity ( K d value of 10–15 nM). Binding of RNA containing 18S (RNA 2 and 3) or 28S (RNA 6) rRNA sequences was not different compared with the control RNAs.

Techniques Used: Binding Assay, Labeling, In Vitro, Polymerase Chain Reaction, Filter-binding Assay, Incubation, Concentration Assay, Positron Emission Tomography, Plasmid Preparation, Construct

3) Product Images from "Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli †"

Article Title: Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli †

Journal:

doi: 10.1128/JB.01566-05

Increasing glycogen phosphorylase activity leads to a marked reduction of glycogen levels in E. coli . (A) Time course analyses of the glycogen content in cells transformed with either pET-15b (▵) or pET-glgP (○) cultured in liquid M9 minimal
Figure Legend Snippet: Increasing glycogen phosphorylase activity leads to a marked reduction of glycogen levels in E. coli . (A) Time course analyses of the glycogen content in cells transformed with either pET-15b (▵) or pET-glgP (○) cultured in liquid M9 minimal

Techniques Used: Activity Assay, Transformation Assay, Positron Emission Tomography, Cell Culture

4) Product Images from "In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins"

Article Title: In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-12-63

Cloning and purification of GST-GFP. A : Schematic of vector construction. (1) A spacer was cloned into the pET 15b vector/plasmid in proximity to the hexa-histadine tag and thrombin site, pJB-HTS (2) This fragment was cloned from the pET 15b vector (3) into the pGex-6p-1 vector to add a GST upstream, pJB-GST-HTS. (4) Red shifted GFP was isolated by primer extension with a spacer and a second hexa-histadine tag and (6) cloned into pJB-GST-HTS yielding pJB-GST-HTS-HS-GFP. B : Gel electrophoresis screening of DNA prepared from colonies following step 1—pJB-HTS—were digested with BglII/HindIII (top) the loss of a 500 bp band (lane 1) and appearance of a 318 bp digestion fragment indicates positive colony for spacer insertion. Arrows indicates 500 bp and 250 bp bands on marker. DNA was digested with NcoI (bottom) to screen for positive insertions. One was chosen for sequencing and further cloning (asterisk). C : Basal GFP expression of a pJB-GST-HTS-HS-GFP containing colony is observed microscopically (brightfield overlaid with epifluorescence) with the scale bar indicating 100 μm. After induction, bacteria express significant green color under UV illumination (bottom) indicating high levels of GFP expression. D : GST-GFP electrophoresed as predicted for the known molecular weight (2) before and (3) after cleavage with thrombin as compared to the (1) molecular weight ladder with arrows indicating 55 kDa, 35 kDa, 25 kDa, respectively.
Figure Legend Snippet: Cloning and purification of GST-GFP. A : Schematic of vector construction. (1) A spacer was cloned into the pET 15b vector/plasmid in proximity to the hexa-histadine tag and thrombin site, pJB-HTS (2) This fragment was cloned from the pET 15b vector (3) into the pGex-6p-1 vector to add a GST upstream, pJB-GST-HTS. (4) Red shifted GFP was isolated by primer extension with a spacer and a second hexa-histadine tag and (6) cloned into pJB-GST-HTS yielding pJB-GST-HTS-HS-GFP. B : Gel electrophoresis screening of DNA prepared from colonies following step 1—pJB-HTS—were digested with BglII/HindIII (top) the loss of a 500 bp band (lane 1) and appearance of a 318 bp digestion fragment indicates positive colony for spacer insertion. Arrows indicates 500 bp and 250 bp bands on marker. DNA was digested with NcoI (bottom) to screen for positive insertions. One was chosen for sequencing and further cloning (asterisk). C : Basal GFP expression of a pJB-GST-HTS-HS-GFP containing colony is observed microscopically (brightfield overlaid with epifluorescence) with the scale bar indicating 100 μm. After induction, bacteria express significant green color under UV illumination (bottom) indicating high levels of GFP expression. D : GST-GFP electrophoresed as predicted for the known molecular weight (2) before and (3) after cleavage with thrombin as compared to the (1) molecular weight ladder with arrows indicating 55 kDa, 35 kDa, 25 kDa, respectively.

Techniques Used: Clone Assay, Purification, Plasmid Preparation, Positron Emission Tomography, Isolation, Nucleic Acid Electrophoresis, Marker, Sequencing, Expressing, Molecular Weight

5) Product Images from "Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen"

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen

Journal: BMC Immunology

doi: 10.1186/1471-2172-8-4

SDS-PAGE and Western Blot Analysis of Recombinant DEK. ( A ) Bacterial lysates and Ni-NTA column purified protein were diluted in reducing sample loading buffer, resolved by SDS-PAGE and stained with Coomassie blue. Lanes were loaded with 1) molecular weight (mw) marker, 2) control bacterial lysate, 10 μl, 3) pET-15b/DEK lysate, 10 μl, 4) 1.5 μg Ni-NTA column-purified DEK, 5) pET-15b/EGFP lysate, 10 μl, 6) 1.5 μg Ni-NTA column-purified EGFP. ( B ) Proteins were resolved by SDS-PAGE and then transferred to PVDF membrane using a Bis-Tris electrophoresis buffer system for western blot analysis. In blot (a) lanes contained: 1) mw marker, 2) control bacterial lysate, 3) pET-15b/DEK lysate, 10 μl, 4) 0.15 μg Ni-NTA column-purified DEK, 5) pET-15b/EGFP lysate, 10 μl, 6) 0.15 μg Ni-NTA column-purified EGFP. Blot (a) was probed with anti-6x-His antibody. For blot (b) a separate gel containing the same samples loaded in blot (a) was probed with anti-human DEK monoclonal antibody. Bound antibody was detected in both blots with alkaline phosphatase (AP)-conjugated rabbit anti-mouse IgG (H+L) secondary antibody, as described in Methods. Data is representative of more than three separate experiments.
Figure Legend Snippet: SDS-PAGE and Western Blot Analysis of Recombinant DEK. ( A ) Bacterial lysates and Ni-NTA column purified protein were diluted in reducing sample loading buffer, resolved by SDS-PAGE and stained with Coomassie blue. Lanes were loaded with 1) molecular weight (mw) marker, 2) control bacterial lysate, 10 μl, 3) pET-15b/DEK lysate, 10 μl, 4) 1.5 μg Ni-NTA column-purified DEK, 5) pET-15b/EGFP lysate, 10 μl, 6) 1.5 μg Ni-NTA column-purified EGFP. ( B ) Proteins were resolved by SDS-PAGE and then transferred to PVDF membrane using a Bis-Tris electrophoresis buffer system for western blot analysis. In blot (a) lanes contained: 1) mw marker, 2) control bacterial lysate, 3) pET-15b/DEK lysate, 10 μl, 4) 0.15 μg Ni-NTA column-purified DEK, 5) pET-15b/EGFP lysate, 10 μl, 6) 0.15 μg Ni-NTA column-purified EGFP. Blot (a) was probed with anti-6x-His antibody. For blot (b) a separate gel containing the same samples loaded in blot (a) was probed with anti-human DEK monoclonal antibody. Bound antibody was detected in both blots with alkaline phosphatase (AP)-conjugated rabbit anti-mouse IgG (H+L) secondary antibody, as described in Methods. Data is representative of more than three separate experiments.

Techniques Used: SDS Page, Western Blot, Recombinant, Purification, Staining, Molecular Weight, Marker, Positron Emission Tomography, Electrophoresis

Related Articles

Clone Assay:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: PCR-amplified DNA fragments were cloned into the pCR2.1-TOPO vector to generate pCR-ppx , pCR-Nppx , pCR-N303ppx , and pCR-Cppx , respectively. .. For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively.

Article Title: A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1
Article Snippet: .. His-Crabp1 was cloned into pET-15b (69665, Millipore Sigma) to generate N-terminal, His6x-tag. .. For His-cRaf-(R) fragment, Raf1 aa 51–187 was cloned into a pET-32a vector (69015, Millipore Sigma).

Article Title: A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution
Article Snippet: .. DNA fragments spanning residues 273–900 encoding the catalytic domains of the DENV RdRp from the four serotypes (DENV 1–4) were amplified by PCR and cloned into pET-15b (Novagen, Madison, USA) using the forward and reverse primers listed in Table 1 . .. Transformed Escherichia coli BL21 (DE3) cells (RIL) (Stratagene) were grown at 310 K in LB medium containing 100 µg ml−1 ampicillin and 50 µg ml−1 chloramphenicol until an OD600 of 0.4–0.6 was attained.

Article Title: Ribonuclease A Homologues of the Zebrafish: Polymorphism, Crystal Structures of Two Representatives and their Evolutionary Implications
Article Snippet: An RNase ZF-1a expression plasmid was created by use of a ligation-independent cloning PCR approach. .. For expression of RNase ZF-3e, the gene was excised from pGEM-z3e with NdeI and XhoI and ligated with similarly treated pET-15b (Novagen) to form pET15-z3e.

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: The coding part of the Ct-RBD-1 gene was cloned into the pET-15b expression vector (Novagen, Madison, WI) and expressed in Escherichia coli . .. To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro.

Article Title: Cnu, a Novel oriC-Binding Protein of Escherichia coli
Article Snippet: .. The cnu gene was PCR amplified from pHL355-36 and cloned between the NdeI and BamHI sites of pET-15b (Novagen), generating pETCnu. .. For the expression of 6×His-Cnu (Cnu protein tagged with six histidines at the N terminus), BL21 (DE3)/pETCnu cells were grown in M9 minimal medium containing Amp (50 mg/ml) at 37°C for 16 h. A one-tenth dilution of this culture was made in fresh M9 medium (1 liter), and cells were grown to an A 600 of 0.7 at 37°C.

Article Title: Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli †
Article Snippet: .. 2.5-kb PCR product thus obtained was cloned in the pGEM-T Easy vector (Promega) to create pG-glgP. glgP was subsequently cloned into either pET-15b (Novagen) or pBAD-18 (a pBAD/His derivative; Invitrogen) expression vectors , as illustrated in Fig. S1 and S2 in the supplemental material, to produce pET-glgP and pBAD-glgP, respectively. ..

Article Title: In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins
Article Snippet: .. The vector for protein production, pJB-GST-HTS-HS-GFP, was created by first cloning a 7 amino acid spacer fragment containing an XhoI site into pET-15b (Novagen) using sticky ligation and BamHI/XhoI sites. .. Colonies were mini-prepped by conventional SDS-precipitation and screened by BglII/HindIII (New England Biolabs; NEB) digestion (Figure B, lower panel), and confirmed by subsequent sequencing (ACGT).

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: Expression of DEK protein Full-length DEK (GeneBank, BC055451 ) was cloned from AGN2a cDNA using the following primers: (fwd) 5'-GGAATTCCATATGCCGGGTCCCAGGGAAGAG, (rev) 5'-CGGGATCCTCAAGAAATTAGCTCTTTTACAG. .. NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing.

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: .. The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen). .. The nucleotide sequence was determined by the dideoxynucleotide chain termination method using T7 sequenase kit (Amersham).

Centrifugation:

Article Title: A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution
Article Snippet: DNA fragments spanning residues 273–900 encoding the catalytic domains of the DENV RdRp from the four serotypes (DENV 1–4) were amplified by PCR and cloned into pET-15b (Novagen, Madison, USA) using the forward and reverse primers listed in Table 1 . .. After overnight growth, cells were harvested by centrifugation at 8000 g for 10 min at 277 K. Pellets were resuspended in buffer A (20 m M Tris–HCl pH 7.5, 0.5 M NaCl, 10 m M β-mercaptoethanol, 10% glycerol) supplemented with an EDTA-free protease-inhibitor tablet (Roche, Switzerland).

Amplification:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: .. For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively. .. These plasmids were then transformed into E. coli BL21-CodonPlus.

Article Title: A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution
Article Snippet: .. DNA fragments spanning residues 273–900 encoding the catalytic domains of the DENV RdRp from the four serotypes (DENV 1–4) were amplified by PCR and cloned into pET-15b (Novagen, Madison, USA) using the forward and reverse primers listed in Table 1 . .. Transformed Escherichia coli BL21 (DE3) cells (RIL) (Stratagene) were grown at 310 K in LB medium containing 100 µg ml−1 ampicillin and 50 µg ml−1 chloramphenicol until an OD600 of 0.4–0.6 was attained.

Article Title: Ribonuclease A Homologues of the Zebrafish: Polymorphism, Crystal Structures of Two Representatives and their Evolutionary Implications
Article Snippet: Construction of expression strains The coding sequences of mature RNases ZF-1a and-3e were amplified from the aforementioned cDNA clones by PCR with KOD Hot Start DNA polymerase (Novagen) in conjunction with the following primers: for RNase ZF-1a 5′-CATATGCAACCTGAACATGTAAAGGAGC-3′ (forward) 5′-CTCGAGTCAGCCTACGTTAACTTCGTCT-3′ (reverse) for RNase ZF-3e 5′-CATATGCAACCAGCAGAAATAAGGCG-3′ (forward) 5′-CTCGAGCTAAACAATAACACCTCTTTCATAGTG-3′ (reverse) Amplification products were A-tailed with Taq polymerase/dATP and ligated with EcoRV-cleaved, T-tailed pGEM®-T Easy Vector (all Promega) to form pGEM-z1a and pGEM-z3e, respectively. .. For expression of RNase ZF-3e, the gene was excised from pGEM-z3e with NdeI and XhoI and ligated with similarly treated pET-15b (Novagen) to form pET15-z3e.

Article Title: Filamin B represses chondrocyte hypertrophy in a Runx2/Smad3-dependent manner
Article Snippet: To produce his-tagged Smad3 protein, PCR-amplified Smad3 gene was subcloned into NdeI and BamHI sites of a bacterial expression vector, pET-15b (Novagen). .. To produce GST fusion protein, the C terminus of Filamin B was PCR amplified and subcloned into NdeI and HindIII sites of a pGEX2TL.

Article Title: Cnu, a Novel oriC-Binding Protein of Escherichia coli
Article Snippet: .. The cnu gene was PCR amplified from pHL355-36 and cloned between the NdeI and BamHI sites of pET-15b (Novagen), generating pETCnu. .. For the expression of 6×His-Cnu (Cnu protein tagged with six histidines at the N terminus), BL21 (DE3)/pETCnu cells were grown in M9 minimal medium containing Amp (50 mg/ml) at 37°C for 16 h. A one-tenth dilution of this culture was made in fresh M9 medium (1 liter), and cells were grown to an A 600 of 0.7 at 37°C.

Article Title: Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli †
Article Snippet: glgP was amplified by PCR using the chromosomal DNA of E. coli BL21(DE3)C43 as template and the following primers: forward, 5′-GCTAGCAGGAGCTCGAGTCCATGAATGCTCCGTTTACATATTC-3′; reverse, 5′-GGATCCTTACAATCTCACCGGATCGATATGC-3′. .. 2.5-kb PCR product thus obtained was cloned in the pGEM-T Easy vector (Promega) to create pG-glgP. glgP was subsequently cloned into either pET-15b (Novagen) or pBAD-18 (a pBAD/His derivative; Invitrogen) expression vectors , as illustrated in Fig. S1 and S2 in the supplemental material, to produce pET-glgP and pBAD-glgP, respectively.

Article Title: Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker
Article Snippet: The DNA fragment, containing two Sap I sites between PCNA2 and PdX, was amplified from the resulting plasmid by PCR using two primers, 5’-TATTTCCAGGGCCATATGATGAAAGCCAAAGTGATCG-3’ (forward) and 5’-ATGGCCCTGGAAATACAGG-3’ (reverse). .. A modified pET-15b(+) (Novagen, Darmstadt, Germany), termed pET15T, which lacked a Sap I site and had a TEV protease recognition site between a thrombin recognition site and an Nde I site, was linearized by PCR using two primers, 5’-GGATCCGGCTGCTAACAAAG-3’ (forward) and 5’-TTAGCAGCCGGATCCTTACCATTGCCTATCGGG-3’ (reverse).

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: The complete ORF cDNA was amplified with a 5′-end sense primer containing a Nde I site (SK30, 5′-AGAGAGTTCCATATGTCTGCAAATTCCAAAATGAACCATAAAAGAGACCAGC-3′) and a 3′-end antisense primer containing a Xho I site (SK31, 5′-GAGAGAGATCTCGAGCTAGTTGGTCTTCTCCAGAGACCTGTAGTAATCTGTCAACAC-3′) using Expand (Boehringer-Mannheim). .. The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen).

Synthesized:

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: The first strand was synthesized by SuperScript II (GIBCO/BRL) using an antisense primer (SK5, 5′-GCAATTGGATACAGAGCAGC-3′) complementary to the 3′-untranslated region of SK2 mRNA. .. The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen).

Construct:

Article Title: A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1
Article Snippet: Paragraph title: Cell Culture and plasmid constructs and transfection ... His-Crabp1 was cloned into pET-15b (69665, Millipore Sigma) to generate N-terminal, His6x-tag.

Article Title: Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker
Article Snippet: Plasmid construction The plasmids expressing PCNA2 protein fused to PdX were constructed by inserting DNA sequences encoding peptide linkers between the C-terminus of PCNA2 and the N-terminus of the Cys73Ser/Cys85Ser mutant of PdX. .. A modified pET-15b(+) (Novagen, Darmstadt, Germany), termed pET15T, which lacked a Sap I site and had a TEV protease recognition site between a thrombin recognition site and an Nde I site, was linearized by PCR using two primers, 5’-GGATCCGGCTGCTAACAAAG-3’ (forward) and 5’-TTAGCAGCCGGATCCTTACCATTGCCTATCGGG-3’ (reverse).

Electrophoresis:

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Bacterial lysates were lysed in reducing loading buffer (NuPAGE system, Invitrogen), proteins resolved by SDS-PAGE, and the proteins transferred to PVDF membranes (Invitrolon, 0.45 μm, Invitrogen) using a NuPAGE Bis-Tris electrophoresis system (Invitrogen).

Incubation:

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro. .. Then 2–3 fmol of RNA (in molecules) was heated at 60°C in 20 mM Tris-HCl, pH 7.5, 200 mM KCl, 5 mM MgCl2 for 15 min, cooled to 20°C, and incubated with different concentrations of purified protein in 60 μl of binding buffer (25 mM Tris-HCl, pH 7.5, 200 mM KCl, 5 mM MgCl2 , 20% glycerol, 50 μg/ml tRNA, 10 μg/ml bovine serum albumin) for 30 min at 20°C.

Activity Assay:

Article Title: Ribonuclease A Homologues of the Zebrafish: Polymorphism, Crystal Structures of Two Representatives and their Evolutionary Implications
Article Snippet: For expression of RNase ZF-3e, the gene was excised from pGEM-z3e with NdeI and XhoI and ligated with similarly treated pET-15b (Novagen) to form pET15-z3e. .. For assay of ribonucleolytic activity, each protein was expressed in Met(–1) form, without a His tag.

Expressing:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: .. For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively. .. These plasmids were then transformed into E. coli BL21-CodonPlus.

Article Title: A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1
Article Snippet: His-Crabp1 was cloned into pET-15b (69665, Millipore Sigma) to generate N-terminal, His6x-tag. .. Flag-HA-Crabp1 expression lentivirus construct was cloned into pCDH-EF1α-MCS-IRES-puro (System Biosciences) and prepared as described .

Article Title: A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution
Article Snippet: Paragraph title: 2.1. Protein expression and purification ... DNA fragments spanning residues 273–900 encoding the catalytic domains of the DENV RdRp from the four serotypes (DENV 1–4) were amplified by PCR and cloned into pET-15b (Novagen, Madison, USA) using the forward and reverse primers listed in Table 1 .

Article Title: Ribonuclease A Homologues of the Zebrafish: Polymorphism, Crystal Structures of Two Representatives and their Evolutionary Implications
Article Snippet: .. For expression of RNase ZF-3e, the gene was excised from pGEM-z3e with NdeI and XhoI and ligated with similarly treated pET-15b (Novagen) to form pET15-z3e. ..

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: The coding part of the Ct-RBD-1 gene was cloned into the pET-15b expression vector (Novagen, Madison, WI) and expressed in Escherichia coli . .. To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro.

Article Title: Filamin B represses chondrocyte hypertrophy in a Runx2/Smad3-dependent manner
Article Snippet: .. To produce his-tagged Smad3 protein, PCR-amplified Smad3 gene was subcloned into NdeI and BamHI sites of a bacterial expression vector, pET-15b (Novagen). .. To produce GST fusion protein, the C terminus of Filamin B was PCR amplified and subcloned into NdeI and HindIII sites of a pGEX2TL.

Article Title: Cnu, a Novel oriC-Binding Protein of Escherichia coli
Article Snippet: Paragraph title: Expression and purification of Cnu and H-NS proteins. ... The cnu gene was PCR amplified from pHL355-36 and cloned between the NdeI and BamHI sites of pET-15b (Novagen), generating pETCnu.

Article Title: Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli †
Article Snippet: .. 2.5-kb PCR product thus obtained was cloned in the pGEM-T Easy vector (Promega) to create pG-glgP. glgP was subsequently cloned into either pET-15b (Novagen) or pBAD-18 (a pBAD/His derivative; Invitrogen) expression vectors , as illustrated in Fig. S1 and S2 in the supplemental material, to produce pET-glgP and pBAD-glgP, respectively. ..

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: Paragraph title: Expression of DEK protein ... NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing.

Article Title: Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker
Article Snippet: Plasmid construction The plasmids expressing PCNA2 protein fused to PdX were constructed by inserting DNA sequences encoding peptide linkers between the C-terminus of PCNA2 and the N-terminus of the Cys73Ser/Cys85Ser mutant of PdX. .. A modified pET-15b(+) (Novagen, Darmstadt, Germany), termed pET15T, which lacked a Sap I site and had a TEV protease recognition site between a thrombin recognition site and an Nde I site, was linearized by PCR using two primers, 5’-GGATCCGGCTGCTAACAAAG-3’ (forward) and 5’-TTAGCAGCCGGATCCTTACCATTGCCTATCGGG-3’ (reverse).

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: .. The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen). .. The nucleotide sequence was determined by the dideoxynucleotide chain termination method using T7 sequenase kit (Amersham).

Modification:

Article Title: Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker
Article Snippet: .. A modified pET-15b(+) (Novagen, Darmstadt, Germany), termed pET15T, which lacked a Sap I site and had a TEV protease recognition site between a thrombin recognition site and an Nde I site, was linearized by PCR using two primers, 5’-GGATCCGGCTGCTAACAAAG-3’ (forward) and 5’-TTAGCAGCCGGATCCTTACCATTGCCTATCGGG-3’ (reverse). .. The amplified DNA fragment was fused to the linearized plasmid using the In-Fusion enzyme (Clontech, Mountain View, CA, USA), to obtain p2SSX.

Western Blot:

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Prokaryotically-expressed proteins were purified using a Ni-NTA Purification System (Invitrogen) and analyzed by SDS-PAGE and western blotting.

Transformation Assay:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: These plasmids were later transformed into E. coli XL10-Gold, followed by selection of ampicillin-resistant transformants. .. For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively.

Article Title: A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution
Article Snippet: DNA fragments spanning residues 273–900 encoding the catalytic domains of the DENV RdRp from the four serotypes (DENV 1–4) were amplified by PCR and cloned into pET-15b (Novagen, Madison, USA) using the forward and reverse primers listed in Table 1 . .. Transformed Escherichia coli BL21 (DE3) cells (RIL) (Stratagene) were grown at 310 K in LB medium containing 100 µg ml−1 ampicillin and 50 µg ml−1 chloramphenicol until an OD600 of 0.4–0.6 was attained.

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Plasmids were transformed into E. coli strain BL21 (DE3) and gene expression induced with 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Invitrogen).

Transfection:

Article Title: A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1
Article Snippet: Paragraph title: Cell Culture and plasmid constructs and transfection ... His-Crabp1 was cloned into pET-15b (69665, Millipore Sigma) to generate N-terminal, His6x-tag.

Sequencing:

Article Title: Ribonuclease A Homologues of the Zebrafish: Polymorphism, Crystal Structures of Two Representatives and their Evolutionary Implications
Article Snippet: For expression of RNase ZF-3e, the gene was excised from pGEM-z3e with NdeI and XhoI and ligated with similarly treated pET-15b (Novagen) to form pET15-z3e. .. The sequence encoding RNase ZF-1a was PCR-amplified from pYSBLIC-z1a with the primers 5′-CATCACCACCACCATATGCAACCTGAACATGTAAAG-3′ (forward) 5′-GGAGAACTCGAGTTAGCCTACGTTAACTTCGTC-3′ (reverse) and treated with NdeI and XhoI, while that encoding RNase ZF-3e was excised from pET15-z3e using the same enzymes.

Article Title: In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins
Article Snippet: The vector for protein production, pJB-GST-HTS-HS-GFP, was created by first cloning a 7 amino acid spacer fragment containing an XhoI site into pET-15b (Novagen) using sticky ligation and BamHI/XhoI sites. .. Colonies were mini-prepped by conventional SDS-precipitation and screened by BglII/HindIII (New England Biolabs; NEB) digestion (Figure B, lower panel), and confirmed by subsequent sequencing (ACGT).

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: .. NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Plasmids were transformed into E. coli strain BL21 (DE3) and gene expression induced with 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Invitrogen).

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: The 5′-end of a cDNA sequence subsequently was obtained by the 5′-inverse PCR method ( ). .. The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen).

Ligation:

Article Title: Ribonuclease A Homologues of the Zebrafish: Polymorphism, Crystal Structures of Two Representatives and their Evolutionary Implications
Article Snippet: An RNase ZF-1a expression plasmid was created by use of a ligation-independent cloning PCR approach. .. For expression of RNase ZF-3e, the gene was excised from pGEM-z3e with NdeI and XhoI and ligated with similarly treated pET-15b (Novagen) to form pET15-z3e.

Article Title: In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins
Article Snippet: .. The vector for protein production, pJB-GST-HTS-HS-GFP, was created by first cloning a 7 amino acid spacer fragment containing an XhoI site into pET-15b (Novagen) using sticky ligation and BamHI/XhoI sites. .. Colonies were mini-prepped by conventional SDS-precipitation and screened by BglII/HindIII (New England Biolabs; NEB) digestion (Figure B, lower panel), and confirmed by subsequent sequencing (ACGT).

Protease Inhibitor:

Article Title: A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution
Article Snippet: DNA fragments spanning residues 273–900 encoding the catalytic domains of the DENV RdRp from the four serotypes (DENV 1–4) were amplified by PCR and cloned into pET-15b (Novagen, Madison, USA) using the forward and reverse primers listed in Table 1 . .. After overnight growth, cells were harvested by centrifugation at 8000 g for 10 min at 277 K. Pellets were resuspended in buffer A (20 m M Tris–HCl pH 7.5, 0.5 M NaCl, 10 m M β-mercaptoethanol, 10% glycerol) supplemented with an EDTA-free protease-inhibitor tablet (Roche, Switzerland).

Cell Culture:

Article Title: A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1
Article Snippet: Paragraph title: Cell Culture and plasmid constructs and transfection ... His-Crabp1 was cloned into pET-15b (69665, Millipore Sigma) to generate N-terminal, His6x-tag.

Generated:

Article Title: Filamin B represses chondrocyte hypertrophy in a Runx2/Smad3-dependent manner
Article Snippet: To generate myc-tagged N terminus–truncated mammalian-expressing Filamin B, the C-terminal 231-amino-acid coding region was generated by PCR and inserted into the StuI–XbaI sites of the CS2+MT vector. .. To produce his-tagged Smad3 protein, PCR-amplified Smad3 gene was subcloned into NdeI and BamHI sites of a bacterial expression vector, pET-15b (Novagen).

DNA Sequencing:

Article Title: Ribonuclease A Homologues of the Zebrafish: Polymorphism, Crystal Structures of Two Representatives and their Evolutionary Implications
Article Snippet: The mixture was then used to transform Escherichia coli JM109 cells and authentic clones were identified by restriction digestion and DNA sequencing. .. For expression of RNase ZF-3e, the gene was excised from pGEM-z3e with NdeI and XhoI and ligated with similarly treated pET-15b (Novagen) to form pET15-z3e.

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: .. NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Plasmids were transformed into E. coli strain BL21 (DE3) and gene expression induced with 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Invitrogen).

Reverse Transcription Polymerase Chain Reaction:

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: RT-PCR of the total liver RNA yielded a single band approximately 1.9 kb in length. .. The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen).

Affinity Purification:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively. .. Affinity purification on Ni-agarose columns was used to perform protein purification, following the manufacturer's protocol (the QIA expressionist , Qiagen).

Binding Assay:

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: Paragraph title: RNA-Protein Binding ... To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro.

Mutagenesis:

Article Title: A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1
Article Snippet: Mutation (G12V) in Hras was introduced with site-directed mutagenesis kit (Agilent Technologies, Cedar Creek, TX). .. His-Crabp1 was cloned into pET-15b (69665, Millipore Sigma) to generate N-terminal, His6x-tag.

Article Title: Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker
Article Snippet: First, two Sap I sites were inserted by PCR between the genes encoding PCNA2 and the Cys73Ser/Cys85Ser mutant of PdX in pHSG+PCNA2-PdX [ ], using two primers, 5’-GGATCCGCTCTTCAGGAGGTGGTGGCTCTATGTCTAAAG-3’ (forward) and 5’-GAATTCGCTCTTCTACCGCCGTCCGCGC-3’ (reverse). .. A modified pET-15b(+) (Novagen, Darmstadt, Germany), termed pET15T, which lacked a Sap I site and had a TEV protease recognition site between a thrombin recognition site and an Nde I site, was linearized by PCR using two primers, 5’-GGATCCGGCTGCTAACAAAG-3’ (forward) and 5’-TTAGCAGCCGGATCCTTACCATTGCCTATCGGG-3’ (reverse).

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen). .. To confirm the bm SK2 mutation at nucleotide 235, genomic DNA sequence at the nucleotide 235 site from the normal and bm mice was determined after amplifying genomic DNA fragments by PCR using a set of two primers flanking the mutation site (SK15, 5′-CAGTAGATCTCGAGTGCGGATATTCTCTTCTCGGTCC-3′; SK26, 5′-CAACAATAAGCTTTCCTTTGGAAGAGTACC-3′).

Labeling:

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro. .. Binding of Ct-RBD-1 to labeled RNA was investigated by a filter-binding assay, essentially as described by .

Purification:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: Paragraph title: 2.4. Expression and Purification of pa Ppx Variants ... For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively.

Article Title: A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution
Article Snippet: Paragraph title: 2.1. Protein expression and purification ... DNA fragments spanning residues 273–900 encoding the catalytic domains of the DENV RdRp from the four serotypes (DENV 1–4) were amplified by PCR and cloned into pET-15b (Novagen, Madison, USA) using the forward and reverse primers listed in Table 1 .

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro. .. The RNA was purified on polyacrylamide gels.

Article Title: Cnu, a Novel oriC-Binding Protein of Escherichia coli
Article Snippet: Paragraph title: Expression and purification of Cnu and H-NS proteins. ... The cnu gene was PCR amplified from pHL355-36 and cloned between the NdeI and BamHI sites of pET-15b (Novagen), generating pETCnu.

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Prokaryotically-expressed proteins were purified using a Ni-NTA Purification System (Invitrogen) and analyzed by SDS-PAGE and western blotting.

Protein Purification:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively. .. Affinity purification on Ni-agarose columns was used to perform protein purification, following the manufacturer's protocol (the QIA expressionist , Qiagen).

Polymerase Chain Reaction:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: PCR-amplified DNA fragments were cloned into the pCR2.1-TOPO vector to generate pCR-ppx , pCR-Nppx , pCR-N303ppx , and pCR-Cppx , respectively. .. For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively.

Article Title: A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution
Article Snippet: .. DNA fragments spanning residues 273–900 encoding the catalytic domains of the DENV RdRp from the four serotypes (DENV 1–4) were amplified by PCR and cloned into pET-15b (Novagen, Madison, USA) using the forward and reverse primers listed in Table 1 . .. Transformed Escherichia coli BL21 (DE3) cells (RIL) (Stratagene) were grown at 310 K in LB medium containing 100 µg ml−1 ampicillin and 50 µg ml−1 chloramphenicol until an OD600 of 0.4–0.6 was attained.

Article Title: Ribonuclease A Homologues of the Zebrafish: Polymorphism, Crystal Structures of Two Representatives and their Evolutionary Implications
Article Snippet: The gene was first PCR-amplified from pGEM-z1a with the primers 5′-CACCACCACCACATGCAACCTGAACATGTAAAGGAGCG-3′ (forward) 5′-GAGGAGAAGGCGCGTTAGCCTACGTTAACTTCGTCTTCATGATAATGCAC-3′ (reverse) The products were treated with phage T4 DNA polymerase (Novagen)/dATP to generate single-stranded overhangs and annealed with suitably prepared pET-YSBLIC (a pET-28a(+) derivative kindly donated by Dr M.J. Fogg, University of York, UK) to form pYSBLIC-z1a. .. For expression of RNase ZF-3e, the gene was excised from pGEM-z3e with NdeI and XhoI and ligated with similarly treated pET-15b (Novagen) to form pET15-z3e.

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: .. To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro. .. The RNA was purified on polyacrylamide gels.

Article Title: Filamin B represses chondrocyte hypertrophy in a Runx2/Smad3-dependent manner
Article Snippet: .. To produce his-tagged Smad3 protein, PCR-amplified Smad3 gene was subcloned into NdeI and BamHI sites of a bacterial expression vector, pET-15b (Novagen). .. To produce GST fusion protein, the C terminus of Filamin B was PCR amplified and subcloned into NdeI and HindIII sites of a pGEX2TL.

Article Title: Cnu, a Novel oriC-Binding Protein of Escherichia coli
Article Snippet: .. The cnu gene was PCR amplified from pHL355-36 and cloned between the NdeI and BamHI sites of pET-15b (Novagen), generating pETCnu. .. For the expression of 6×His-Cnu (Cnu protein tagged with six histidines at the N terminus), BL21 (DE3)/pETCnu cells were grown in M9 minimal medium containing Amp (50 mg/ml) at 37°C for 16 h. A one-tenth dilution of this culture was made in fresh M9 medium (1 liter), and cells were grown to an A 600 of 0.7 at 37°C.

Article Title: Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli †
Article Snippet: .. 2.5-kb PCR product thus obtained was cloned in the pGEM-T Easy vector (Promega) to create pG-glgP. glgP was subsequently cloned into either pET-15b (Novagen) or pBAD-18 (a pBAD/His derivative; Invitrogen) expression vectors , as illustrated in Fig. S1 and S2 in the supplemental material, to produce pET-glgP and pBAD-glgP, respectively. ..

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: .. NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Plasmids were transformed into E. coli strain BL21 (DE3) and gene expression induced with 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Invitrogen).

Article Title: Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker
Article Snippet: .. A modified pET-15b(+) (Novagen, Darmstadt, Germany), termed pET15T, which lacked a Sap I site and had a TEV protease recognition site between a thrombin recognition site and an Nde I site, was linearized by PCR using two primers, 5’-GGATCCGGCTGCTAACAAAG-3’ (forward) and 5’-TTAGCAGCCGGATCCTTACCATTGCCTATCGGG-3’ (reverse). .. The amplified DNA fragment was fused to the linearized plasmid using the In-Fusion enzyme (Clontech, Mountain View, CA, USA), to obtain p2SSX.

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: The 5′-end of a cDNA sequence subsequently was obtained by the 5′-inverse PCR method ( ). .. The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen).

Selection:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: These plasmids were later transformed into E. coli XL10-Gold, followed by selection of ampicillin-resistant transformants. .. For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively.

Article Title: A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1
Article Snippet: His-Crabp1 was cloned into pET-15b (69665, Millipore Sigma) to generate N-terminal, His6x-tag. .. Stable clones expressing Flag-HA-Crabp1 and empty vector were established in HEK293T cells using puromycin selection and transfected by routine calcium phosphate method for the reconstitution of MAPK signaling components.

Mouse Assay:

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen). .. To confirm the bm SK2 mutation at nucleotide 235, genomic DNA sequence at the nucleotide 235 site from the normal and bm mice was determined after amplifying genomic DNA fragments by PCR using a set of two primers flanking the mutation site (SK15, 5′-CAGTAGATCTCGAGTGCGGATATTCTCTTCTCGGTCC-3′; SK26, 5′-CAACAATAAGCTTTCCTTTGGAAGAGTACC-3′).

SDS Page:

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Prokaryotically-expressed proteins were purified using a Ni-NTA Purification System (Invitrogen) and analyzed by SDS-PAGE and western blotting.

Plasmid Preparation:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: PCR-amplified DNA fragments were cloned into the pCR2.1-TOPO vector to generate pCR-ppx , pCR-Nppx , pCR-N303ppx , and pCR-Cppx , respectively. .. For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively.

Article Title: A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1
Article Snippet: Paragraph title: Cell Culture and plasmid constructs and transfection ... His-Crabp1 was cloned into pET-15b (69665, Millipore Sigma) to generate N-terminal, His6x-tag.

Article Title: Ribonuclease A Homologues of the Zebrafish: Polymorphism, Crystal Structures of Two Representatives and their Evolutionary Implications
Article Snippet: An RNase ZF-1a expression plasmid was created by use of a ligation-independent cloning PCR approach. .. For expression of RNase ZF-3e, the gene was excised from pGEM-z3e with NdeI and XhoI and ligated with similarly treated pET-15b (Novagen) to form pET15-z3e.

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: .. To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro. .. The RNA was purified on polyacrylamide gels.

Article Title: Filamin B represses chondrocyte hypertrophy in a Runx2/Smad3-dependent manner
Article Snippet: .. To produce his-tagged Smad3 protein, PCR-amplified Smad3 gene was subcloned into NdeI and BamHI sites of a bacterial expression vector, pET-15b (Novagen). .. To produce GST fusion protein, the C terminus of Filamin B was PCR amplified and subcloned into NdeI and HindIII sites of a pGEX2TL.

Article Title: Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli †
Article Snippet: .. 2.5-kb PCR product thus obtained was cloned in the pGEM-T Easy vector (Promega) to create pG-glgP. glgP was subsequently cloned into either pET-15b (Novagen) or pBAD-18 (a pBAD/His derivative; Invitrogen) expression vectors , as illustrated in Fig. S1 and S2 in the supplemental material, to produce pET-glgP and pBAD-glgP, respectively. ..

Article Title: In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins
Article Snippet: .. The vector for protein production, pJB-GST-HTS-HS-GFP, was created by first cloning a 7 amino acid spacer fragment containing an XhoI site into pET-15b (Novagen) using sticky ligation and BamHI/XhoI sites. .. Colonies were mini-prepped by conventional SDS-precipitation and screened by BglII/HindIII (New England Biolabs; NEB) digestion (Figure B, lower panel), and confirmed by subsequent sequencing (ACGT).

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: .. NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Plasmids were transformed into E. coli strain BL21 (DE3) and gene expression induced with 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Invitrogen).

Article Title: Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker
Article Snippet: Paragraph title: Plasmid construction ... A modified pET-15b(+) (Novagen, Darmstadt, Germany), termed pET15T, which lacked a Sap I site and had a TEV protease recognition site between a thrombin recognition site and an Nde I site, was linearized by PCR using two primers, 5’-GGATCCGGCTGCTAACAAAG-3’ (forward) and 5’-TTAGCAGCCGGATCCTTACCATTGCCTATCGGG-3’ (reverse).

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: .. The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen). .. The nucleotide sequence was determined by the dideoxynucleotide chain termination method using T7 sequenase kit (Amersham).

Irradiation:

Article Title: A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1
Article Snippet: Cells were grown on irradiated mouse embryonic feeder cells in 0.2% (w/v) gelatin-coated plates. .. His-Crabp1 was cloned into pET-15b (69665, Millipore Sigma) to generate N-terminal, His6x-tag.

Positron Emission Tomography:

Article Title: Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase
Article Snippet: .. For gene expression, the different amplified ppx fragments were restricted by Eco RI-Nde I enzymes and subcloned into pET-15b (Novagen) as N-terminal fusions to a 6xHis-tag, generating pET-ppx , pET-Nppx , pET-N303ppx , and pET-Cppx , respectively. .. These plasmids were then transformed into E. coli BL21-CodonPlus.

Article Title: A new regulatory mechanism for Raf kinase activation, retinoic acid-bound Crabp1
Article Snippet: .. His-Crabp1 was cloned into pET-15b (69665, Millipore Sigma) to generate N-terminal, His6x-tag. .. For His-cRaf-(R) fragment, Raf1 aa 51–187 was cloned into a pET-32a vector (69015, Millipore Sigma).

Article Title: A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution
Article Snippet: .. DNA fragments spanning residues 273–900 encoding the catalytic domains of the DENV RdRp from the four serotypes (DENV 1–4) were amplified by PCR and cloned into pET-15b (Novagen, Madison, USA) using the forward and reverse primers listed in Table 1 . .. Transformed Escherichia coli BL21 (DE3) cells (RIL) (Stratagene) were grown at 310 K in LB medium containing 100 µg ml−1 ampicillin and 50 µg ml−1 chloramphenicol until an OD600 of 0.4–0.6 was attained.

Article Title: Ribonuclease A Homologues of the Zebrafish: Polymorphism, Crystal Structures of Two Representatives and their Evolutionary Implications
Article Snippet: .. For expression of RNase ZF-3e, the gene was excised from pGEM-z3e with NdeI and XhoI and ligated with similarly treated pET-15b (Novagen) to form pET15-z3e. ..

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: .. To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro. .. The RNA was purified on polyacrylamide gels.

Article Title: Filamin B represses chondrocyte hypertrophy in a Runx2/Smad3-dependent manner
Article Snippet: .. To produce his-tagged Smad3 protein, PCR-amplified Smad3 gene was subcloned into NdeI and BamHI sites of a bacterial expression vector, pET-15b (Novagen). .. To produce GST fusion protein, the C terminus of Filamin B was PCR amplified and subcloned into NdeI and HindIII sites of a pGEX2TL.

Article Title: Cnu, a Novel oriC-Binding Protein of Escherichia coli
Article Snippet: .. The cnu gene was PCR amplified from pHL355-36 and cloned between the NdeI and BamHI sites of pET-15b (Novagen), generating pETCnu. .. For the expression of 6×His-Cnu (Cnu protein tagged with six histidines at the N terminus), BL21 (DE3)/pETCnu cells were grown in M9 minimal medium containing Amp (50 mg/ml) at 37°C for 16 h. A one-tenth dilution of this culture was made in fresh M9 medium (1 liter), and cells were grown to an A 600 of 0.7 at 37°C.

Article Title: Glycogen Phosphorylase, the Product of the glgP Gene, Catalyzes Glycogen Breakdown by Removing Glucose Units from the Nonreducing Ends in Escherichia coli †
Article Snippet: .. 2.5-kb PCR product thus obtained was cloned in the pGEM-T Easy vector (Promega) to create pG-glgP. glgP was subsequently cloned into either pET-15b (Novagen) or pBAD-18 (a pBAD/His derivative; Invitrogen) expression vectors , as illustrated in Fig. S1 and S2 in the supplemental material, to produce pET-glgP and pBAD-glgP, respectively. ..

Article Title: In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins
Article Snippet: .. The vector for protein production, pJB-GST-HTS-HS-GFP, was created by first cloning a 7 amino acid spacer fragment containing an XhoI site into pET-15b (Novagen) using sticky ligation and BamHI/XhoI sites. .. Colonies were mini-prepped by conventional SDS-precipitation and screened by BglII/HindIII (New England Biolabs; NEB) digestion (Figure B, lower panel), and confirmed by subsequent sequencing (ACGT).

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: .. NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Plasmids were transformed into E. coli strain BL21 (DE3) and gene expression induced with 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Invitrogen).

Article Title: Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker
Article Snippet: .. A modified pET-15b(+) (Novagen, Darmstadt, Germany), termed pET15T, which lacked a Sap I site and had a TEV protease recognition site between a thrombin recognition site and an Nde I site, was linearized by PCR using two primers, 5’-GGATCCGGCTGCTAACAAAG-3’ (forward) and 5’-TTAGCAGCCGGATCCTTACCATTGCCTATCGGG-3’ (reverse). .. The amplified DNA fragment was fused to the linearized plasmid using the In-Fusion enzyme (Clontech, Mountain View, CA, USA), to obtain p2SSX.

Article Title: A member of a family of sulfate-activating enzymes causes murine brachymorphism
Article Snippet: .. The fragment was digested with Nde I and Xho I and cloned into appropriate restriction sites in a bacterial expression vector, pET-15b (Novagen). .. The nucleotide sequence was determined by the dideoxynucleotide chain termination method using T7 sequenase kit (Amersham).

In Vitro:

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: .. To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro. .. The RNA was purified on polyacrylamide gels.

Affinity Chromatography:

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: His-tagged Ct-RBD-1 was purifed by Ni2+ -NTA affinity chromatography (QIAGEN, Valencia, CA). .. To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro.

Concentration Assay:

Article Title: A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution
Article Snippet: DNA fragments spanning residues 273–900 encoding the catalytic domains of the DENV RdRp from the four serotypes (DENV 1–4) were amplified by PCR and cloned into pET-15b (Novagen, Madison, USA) using the forward and reverse primers listed in Table 1 . .. Protein expression was induced at 289 K by adding isopropyl β- d -thiogalactopyranoside to a final concentration of 0.4 m M .

Article Title: Cnu, a Novel oriC-Binding Protein of Escherichia coli
Article Snippet: The cnu gene was PCR amplified from pHL355-36 and cloned between the NdeI and BamHI sites of pET-15b (Novagen), generating pETCnu. .. IPTG (final concentration, 0.4 mM) was added, and the culture was grown for an additional 6 h. Cells were pelleted and resuspended in 35 ml of phosphate-buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4 , 1.8 mM KH2 PO4 ).

Filter-binding Assay:

Article Title: A Novel Conserved RNA-binding Domain Protein, RBD-1, Is Essential For Ribosome Biogenesis
Article Snippet: To synthesize 32 P-labeled RNA, PCR fragments or linearized plasmid DNA, pET-15b (Novagen), and pBluescript (Stratagene) were transcribed by T7 polymerase in vitro. .. Binding of Ct-RBD-1 to labeled RNA was investigated by a filter-binding assay, essentially as described by .

Recombinant:

Article Title: Serum from mice immunized in the context of Treg inhibition identifies DEK as a neuroblastoma tumor antigen
Article Snippet: .. NdeI and BamHI restricted PCR fragments were ligated into pET-15b (Novagen, Madison, WI) and recombinant plasmid insert sequence (pET-15/DEK and pET-15/EGFP) was verified by DNA sequencing. .. Plasmids were transformed into E. coli strain BL21 (DE3) and gene expression induced with 0.8 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Invitrogen).

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