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Promega shrimp alkaline phosphatase
Shrimp Alkaline Phosphatase, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 87 article reviews
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shrimp alkaline phosphatase - by Bioz Stars, 2020-01
92/100 stars

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Clone Assay:

Article Title: The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines
Article Snippet: The plasmid pET-28a (+) (Novagen) carries an N-terminal His-Tag/thrombin/T7 configuration, and the expression of the cloned gene is under the control of a T7 promoter. .. The linearized plasmid was treated with 2 units of shrimp alkaline phosphatase (Promega, Madison, USA) at 37°C for 30 min.

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: Paragraph title: Cloning ... 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C.

Amplification:

Article Title: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: .. DNA plasmids and Antibodies Mammalian expression vectors pCIG2 (modified to comprise an N-terminal FLAG tag) and pcDNA3 (modified to comprise an N-terminal FLAG or HA epitope tag) were digested with EcoRI and treated with shrimp alkaline phosphatase (Promega, Australia) before ligation to mouse Eurl cDNA amplified by PCR using primers which encoded flanking MfeI restriction sites, together with a template (IMAGE:3966397 also known as D16Ertd472e, Genbank Accession Number BC019957). ..

Article Title: Association of LOX-1 gene polymorphisms with cerebral infarction in northern Chinese Han population
Article Snippet: PCR amplification was performed in a total volume of 10 μL that contained 1× GC buffer I (Takara, Japan), 3.0 mM Mg2+ (Takara, Japan), 0.3 mM dNTP (Generay Biotech, China), 1 U HotStarTaq polymerase (Qiagen, Germany), 1 μL each primers (Sangon, China) and 1 μL genomic DNA. .. The PCR cycling program was set at 95°C for 2 min, followed by 11 cycles of 94°C for 20 s, 65°C (decreased 0.5°C per cycle) for 40 s, 72°C for 1.5 min, and then 24 cycles of 94°C for 20 s, 59°C for 30 s, and 72°C for 1.5 min, and a final extension at 72°C for 2 min. Then shrimp alkaline phosphatase (Promega, USA) and Exonuclease I (Epicenter, USA) were added into the PCR products for purification.

Article Title: Targeted Deep Sequencing Identifies Rare ‘loss-of-function’ Variants in IFNGR1 for Risk of Atopic Dermatitis Complicated by Eczema Herpeticum
Article Snippet: PCR amplification was carried out in 25-μL reactions with 1X Taq Gold Buffer, 0.6 μM dNTPs, 0.125 μM forward and reverse primers, 40 ng of DNA, and 1.25 UTaq Gold polymerase (Life Technologies). .. The reaction was then cycled with the following conditions: initial denaturation at 94°C for 12 min; 40 cycles at 94°C for 20 sec, 54°C for 20 sec, and 72°C for 20 sec; final extension at 72°C for 10 min. PCR products were purified using Exonuclease III and Shrimp Alkaline Phosphatase (Promega, Madison, WI).

Article Title: Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation
Article Snippet: Heavy and light chain DNA of mouse anti-mouse CD20 monoclonal antibody 5D2.16.8 (previously cloned from a hybridoma cell line into mouse IgG2b and mouse kappa) was diluted to less than 500 ng/µl and amplified through PCR with a premix Taq polymerase kit (Takara) using forward primers corresponding to antibody framework region and human vector signal sequence and reverse primers corresponding to antibody framework region and human IgG4 or kappa CH1 (primers shown below). .. Digestion products were purified by gel extraction (Qiagen) and vectors were treated with shrimp alkaline phosphatase (Promega) to prevent self-ligation.

Construct:

Article Title: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: DNA plasmids and Antibodies Mammalian expression vectors pCIG2 (modified to comprise an N-terminal FLAG tag) and pcDNA3 (modified to comprise an N-terminal FLAG or HA epitope tag) were digested with EcoRI and treated with shrimp alkaline phosphatase (Promega, Australia) before ligation to mouse Eurl cDNA amplified by PCR using primers which encoded flanking MfeI restriction sites, together with a template (IMAGE:3966397 also known as D16Ertd472e, Genbank Accession Number BC019957). .. All constructs were sequenced to confirm directionality and nucleotide identity.

Electrophoresis:

Article Title: Targeted Deep Sequencing Identifies Rare ‘loss-of-function’ Variants in IFNGR1 for Risk of Atopic Dermatitis Complicated by Eczema Herpeticum
Article Snippet: The reaction was then cycled with the following conditions: initial denaturation at 94°C for 12 min; 40 cycles at 94°C for 20 sec, 54°C for 20 sec, and 72°C for 20 sec; final extension at 72°C for 10 min. PCR products were purified using Exonuclease III and Shrimp Alkaline Phosphatase (Promega, Madison, WI). .. Automated sequencing was performed by capillary electrophoresis on an ABI3700 (Applied Biosystems) at the Johns Hopkins Genetic Resources Core Facility and all variants were manually called by visual inspection of the electropherogram, using Sequencher software (Gene Codes Corp., Inc.).

Article Title: A Functional Polymorphism in the Promoter Region of MicroRNA-146a Is Associated with the Risk of Alzheimer Disease and the Rate of Cognitive Decline in Patients
Article Snippet: Extension products were purified by a 15-minute incubation with 1U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C and a subsequent 15-minute incubation at 80°C to inactivate the enzyme. .. The purified products (0.5 µl) were mixed with 9 µl of formamide and 0.5 µl of GeneScan-120 LIZ Size Standard (Applied Biosystems) and separated by capillary electrophoresis (ABI PRISM310 Genetic Analyzer; Applied Biosystems).

Incubation:

Article Title: The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines
Article Snippet: The linearized plasmid was treated with 2 units of shrimp alkaline phosphatase (Promega, Madison, USA) at 37°C for 30 min. .. The final volume was adjusted to 10 μL by the addition of nuclease free water, and the tube was incubated at 16°C overnight.

Article Title: The role of the priming loop in Influenza A virus RNA synthesis
Article Snippet: .. The reactions were incubated at 30 °C for 2 h and depending on the experiment then either i) heated to 95 °C for 2 min, treated with 0.5 U shrimp alkaline phosphatase (SAP, Promega) for 30 min at 37 °C, and subsequently stopped with 3 μl stopping solution (90% formamide, 10 mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), or ii) directly stopped with 3 μl stopping solution. ..

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: The plasmid pYUB412 was linearized with XbaI, dephosphorylated with shrimp alkaline phosphatase (Promega), and then digested with BclI to obtain 3,789-bp and 4,773-bp fragments. .. The ligation reaction mixture (5 µl) was mixed with 25 µl of MaxPlax Lambda Packaging Extract (Epicentre, Madison, WI) at room temperature (RT) for 1 h. Another 2 µl of ligation reaction mixture was added to the packaging reaction, and the mixture was incubated for an additional hour at RT.

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C. .. The ligation reaction was performed with 1 μl pG-L4440-ppt-1 added to 3 μl ath-1 , 2 μl 5× T4 ligase buffer, 1 μl T4 DNA ligase and 3 μl H2 O and incubated at room temperature for one hour.

Article Title: Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation
Article Snippet: Digestion products were purified by gel extraction (Qiagen) and vectors were treated with shrimp alkaline phosphatase (Promega) to prevent self-ligation. .. 48 colonies were picked from each of the heavy and light chain transformation plates into 2YT media containing 50 µg/ml carbenicillin and incubated 15 hours at 37°C.

Article Title: Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which includes the loss of tumor suppressor genes involved in the control of cell growth and death
Article Snippet: .. This was followed by a final extension step at 72°C for 10 min. PCR products were purified by incubation with 0.3 U shrimp alkaline phosphatase (Promega, Madison, WI, USA) and 1.5 U exonuclease I (NEB, Ipswich, MA, USA) at 37°C for 8 min, followed by 15 min at 72°C. ..

Article Title: A Functional Polymorphism in the Promoter Region of MicroRNA-146a Is Associated with the Risk of Alzheimer Disease and the Rate of Cognitive Decline in Patients
Article Snippet: .. Extension products were purified by a 15-minute incubation with 1U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C and a subsequent 15-minute incubation at 80°C to inactivate the enzyme. .. The purified products (0.5 µl) were mixed with 9 µl of formamide and 0.5 µl of GeneScan-120 LIZ Size Standard (Applied Biosystems) and separated by capillary electrophoresis (ABI PRISM310 Genetic Analyzer; Applied Biosystems).

Article Title: Promiscuous RNA Binding Ensures Effective Encapsidation of APOBEC3 Proteins by HIV-1
Article Snippet: Tubes were incubated at 37°C for 3 min and added to protein G dynabeads previously incubated with anti-T7 antibody (Novagen) or anti-GFP antibody (Roche). .. The RNAs were dephosphorylated using Shrimp alkaline phosphatase (Promega) and a pre-adenylated linker was ligated to the 3’-end of RNAs on beads.

Activity Assay:

Article Title: The role of the priming loop in Influenza A virus RNA synthesis
Article Snippet: The reactions were incubated at 30 °C for 2 h and depending on the experiment then either i) heated to 95 °C for 2 min, treated with 0.5 U shrimp alkaline phosphatase (SAP, Promega) for 30 min at 37 °C, and subsequently stopped with 3 μl stopping solution (90% formamide, 10 mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), or ii) directly stopped with 3 μl stopping solution. .. To assess ApG synthesis activity, we replaced ATP in the pppApG synthesis reaction with 1 mM adenosine.

Expressing:

Article Title: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: .. DNA plasmids and Antibodies Mammalian expression vectors pCIG2 (modified to comprise an N-terminal FLAG tag) and pcDNA3 (modified to comprise an N-terminal FLAG or HA epitope tag) were digested with EcoRI and treated with shrimp alkaline phosphatase (Promega, Australia) before ligation to mouse Eurl cDNA amplified by PCR using primers which encoded flanking MfeI restriction sites, together with a template (IMAGE:3966397 also known as D16Ertd472e, Genbank Accession Number BC019957). ..

Article Title: The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines
Article Snippet: The plasmid pET-28a (+) (Novagen) carries an N-terminal His-Tag/thrombin/T7 configuration, and the expression of the cloned gene is under the control of a T7 promoter. .. The linearized plasmid was treated with 2 units of shrimp alkaline phosphatase (Promega, Madison, USA) at 37°C for 30 min.

Modification:

Article Title: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: .. DNA plasmids and Antibodies Mammalian expression vectors pCIG2 (modified to comprise an N-terminal FLAG tag) and pcDNA3 (modified to comprise an N-terminal FLAG or HA epitope tag) were digested with EcoRI and treated with shrimp alkaline phosphatase (Promega, Australia) before ligation to mouse Eurl cDNA amplified by PCR using primers which encoded flanking MfeI restriction sites, together with a template (IMAGE:3966397 also known as D16Ertd472e, Genbank Accession Number BC019957). ..

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: The plasmid pYUB412 was linearized with XbaI, dephosphorylated with shrimp alkaline phosphatase (Promega), and then digested with BclI to obtain 3,789-bp and 4,773-bp fragments. .. M. tuberculosis H37Rv genomic DNA was prepared from a 20-ml culture (OD600 , ~3) grown in Middlebrook 7H9 supplemented with 10% OADC, 0.2% glycerol, and 0.05% Tween 80 as previously described , with the following modification: the genomic DNA, once precipitated in isopropanol, was spooled and immersed in ethanol and was then air-dried instead of being spun down.

Article Title: Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which includes the loss of tumor suppressor genes involved in the control of cell growth and death
Article Snippet: PCR reactions were carried out by incubating bisulphite modified DNA with 1.25 U of platinum taq (Invitrogen, Carsbad, CA, USA), 8.3 mM dNTPs, 2 mM MgCl2 and forward and reverse primers (1 μM). .. This was followed by a final extension step at 72°C for 10 min. PCR products were purified by incubation with 0.3 U shrimp alkaline phosphatase (Promega, Madison, WI, USA) and 1.5 U exonuclease I (NEB, Ipswich, MA, USA) at 37°C for 8 min, followed by 15 min at 72°C.

Transformation Assay:

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: The vectors were then transformed into HT115 cells for consistency with the Vidal RNAi library. .. 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C.

Article Title: Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation
Article Snippet: Digestion products were purified by gel extraction (Qiagen) and vectors were treated with shrimp alkaline phosphatase (Promega) to prevent self-ligation. .. 48 colonies were picked from each of the heavy and light chain transformation plates into 2YT media containing 50 µg/ml carbenicillin and incubated 15 hours at 37°C.

Bisulfite Sequencing:

Article Title: Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which includes the loss of tumor suppressor genes involved in the control of cell growth and death
Article Snippet: Paragraph title: Bisulphite sequencing ... This was followed by a final extension step at 72°C for 10 min. PCR products were purified by incubation with 0.3 U shrimp alkaline phosphatase (Promega, Madison, WI, USA) and 1.5 U exonuclease I (NEB, Ipswich, MA, USA) at 37°C for 8 min, followed by 15 min at 72°C.

Ligation:

Article Title: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: .. DNA plasmids and Antibodies Mammalian expression vectors pCIG2 (modified to comprise an N-terminal FLAG tag) and pcDNA3 (modified to comprise an N-terminal FLAG or HA epitope tag) were digested with EcoRI and treated with shrimp alkaline phosphatase (Promega, Australia) before ligation to mouse Eurl cDNA amplified by PCR using primers which encoded flanking MfeI restriction sites, together with a template (IMAGE:3966397 also known as D16Ertd472e, Genbank Accession Number BC019957). ..

Article Title: The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines
Article Snippet: The linearized plasmid was treated with 2 units of shrimp alkaline phosphatase (Promega, Madison, USA) at 37°C for 30 min. .. The ligation reaction was carried out in a tube containing 2 μL (50 ng) of pET28a (+), 2 μL (100 ng) of IFNε cDNA insert, 1 μL 10X ligase buffer, and 1 μL (2 units) of ligase enzyme.

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: The plasmid pYUB412 was linearized with XbaI, dephosphorylated with shrimp alkaline phosphatase (Promega), and then digested with BclI to obtain 3,789-bp and 4,773-bp fragments. .. The ligation reaction mixture (5 µl) was mixed with 25 µl of MaxPlax Lambda Packaging Extract (Epicentre, Madison, WI) at room temperature (RT) for 1 h. Another 2 µl of ligation reaction mixture was added to the packaging reaction, and the mixture was incubated for an additional hour at RT.

Article Title: Association of NLRP1 and NLRP3 Polymorphisms with Psoriasis Vulgaris Risk in the Chinese Han Population
Article Snippet: The products were purified via digestion of 5 U shrimp alkaline phosphatase (Promega) and 2 U exonuclease I at 37°C for 1 h, followed by 75°C for 15 min. .. The ligation reaction was carried out in 10 μ L of a mixture comprising 1 μ L 10x ligation buffer, 0.25 μ L 80 U/μ L Taq DNA ligase (New England Biolabs), 0.4 μ L 5′ ligation primer mixture (1 μ M), 2 μ L purified PCR products, and 6 μ L ddH2O.

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: 20 μl DNA was digested at 37°C in a reaction volume of 25 μl: pG-L4440-ppt-1 was linearised using Acc65I ; pG-L4440-ath-1 was digested with BsrGI to obtain the ath-1 sequence but retaining complementary 5’ overhangs to enable its ligation into the pG-L4440-ppt-1 vector. .. 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C.

Protease Inhibitor:

Article Title: Promiscuous RNA Binding Ensures Effective Encapsidation of APOBEC3 Proteins by HIV-1
Article Snippet: Cells and viruses were then resuspended in 1 ml of lysis buffer (50 mM Tris-HCL, pH 7.4; 100 mM NaCl; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and protease inhibitor) and sonicated. .. The RNAs were dephosphorylated using Shrimp alkaline phosphatase (Promega) and a pre-adenylated linker was ligated to the 3’-end of RNAs on beads.

Mutagenesis:

Article Title: Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which includes the loss of tumor suppressor genes involved in the control of cell growth and death
Article Snippet: This was followed by a final extension step at 72°C for 10 min. PCR products were purified by incubation with 0.3 U shrimp alkaline phosphatase (Promega, Madison, WI, USA) and 1.5 U exonuclease I (NEB, Ipswich, MA, USA) at 37°C for 8 min, followed by 15 min at 72°C. .. Sequences were analyzed using Mutation Surveyor v3.97 (SoftGenetics, PA, USA).

DNA Sequencing:

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: Successful incorporation into the pG-L4440 vector was verified by transforming into DH5α cells and sequencing the purified DNA (DNA Sequencing & Services, University of Dundee, UK). .. 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C.

Article Title: Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species
Article Snippet: Paragraph title: 3.3. DNA Extraction, PCR Primers, Cycling Profiles and DNA Sequencing ... For sequence analysis PCR-amplified DNA fragments were purified with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following program: 30 min at 37 °C and 15 min at 80 °C.

Polymerase Chain Reaction:

Article Title: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: .. DNA plasmids and Antibodies Mammalian expression vectors pCIG2 (modified to comprise an N-terminal FLAG tag) and pcDNA3 (modified to comprise an N-terminal FLAG or HA epitope tag) were digested with EcoRI and treated with shrimp alkaline phosphatase (Promega, Australia) before ligation to mouse Eurl cDNA amplified by PCR using primers which encoded flanking MfeI restriction sites, together with a template (IMAGE:3966397 also known as D16Ertd472e, Genbank Accession Number BC019957). ..

Article Title: Association of LOX-1 gene polymorphisms with cerebral infarction in northern Chinese Han population
Article Snippet: .. The PCR cycling program was set at 95°C for 2 min, followed by 11 cycles of 94°C for 20 s, 65°C (decreased 0.5°C per cycle) for 40 s, 72°C for 1.5 min, and then 24 cycles of 94°C for 20 s, 59°C for 30 s, and 72°C for 1.5 min, and a final extension at 72°C for 2 min. Then shrimp alkaline phosphatase (Promega, USA) and Exonuclease I (Epicenter, USA) were added into the PCR products for purification. ..

Article Title: Association of NLRP1 and NLRP3 Polymorphisms with Psoriasis Vulgaris Risk in the Chinese Han Population
Article Snippet: The PCR cycling program was as follows: 95°C for 2 min; 11 cycles of 94°C for 20 s, decreasing to 65°C at 0.5°C increments per cycle for 40 s, and 72°C for 1.5 min; 24 cycles of 94°C for 20 s, 59°C for 30 s, and 72°C for 1.5 min; and a final extension at 72°C for 2 min, followed by holding at 4°C. .. The products were purified via digestion of 5 U shrimp alkaline phosphatase (Promega) and 2 U exonuclease I at 37°C for 1 h, followed by 75°C for 15 min.

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: Gateway™ cloning was used to shuttle each PCR product into the pG-L4440 vector, with BP clonase™ II and LR clonase™ II (Sigma, Dorset, UK) steps performed as per the manufacturer’s instructions. .. 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C.

Article Title: Targeted Deep Sequencing Identifies Rare ‘loss-of-function’ Variants in IFNGR1 for Risk of Atopic Dermatitis Complicated by Eczema Herpeticum
Article Snippet: .. The reaction was then cycled with the following conditions: initial denaturation at 94°C for 12 min; 40 cycles at 94°C for 20 sec, 54°C for 20 sec, and 72°C for 20 sec; final extension at 72°C for 10 min. PCR products were purified using Exonuclease III and Shrimp Alkaline Phosphatase (Promega, Madison, WI). .. Automated sequencing was performed by capillary electrophoresis on an ABI3700 (Applied Biosystems) at the Johns Hopkins Genetic Resources Core Facility and all variants were manually called by visual inspection of the electropherogram, using Sequencher software (Gene Codes Corp., Inc.).

Article Title: Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation
Article Snippet: The light chain PCR product and pRK human kappa LPG3 vector were digested using KpnI and EcoRI restriction enzymes (NEB) according to manufacturer recommendations. .. Digestion products were purified by gel extraction (Qiagen) and vectors were treated with shrimp alkaline phosphatase (Promega) to prevent self-ligation.

Article Title: Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which includes the loss of tumor suppressor genes involved in the control of cell growth and death
Article Snippet: .. This was followed by a final extension step at 72°C for 10 min. PCR products were purified by incubation with 0.3 U shrimp alkaline phosphatase (Promega, Madison, WI, USA) and 1.5 U exonuclease I (NEB, Ipswich, MA, USA) at 37°C for 8 min, followed by 15 min at 72°C. ..

Article Title: A Functional Polymorphism in the Promoter Region of MicroRNA-146a Is Associated with the Risk of Alzheimer Disease and the Rate of Cognitive Decline in Patients
Article Snippet: Briefly, the SNapShot reaction was carried out in a 10 µl final volume containing SNapShot Multiplex Ready Mix (5 µl), primer mix (0.02–0.6 µmol/L), and templates (4 µl) consisting of the multiplex polymerase chain reaction (PCR) products, which had been purified with the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). .. Extension products were purified by a 15-minute incubation with 1U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C and a subsequent 15-minute incubation at 80°C to inactivate the enzyme.

Article Title: Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species
Article Snippet: .. For sequence analysis PCR-amplified DNA fragments were purified with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following program: 30 min at 37 °C and 15 min at 80 °C. .. Both strands were labeled using a BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to Błaszczyk et al. [ ], and precipitated with ethanol.

Sonication:

Article Title: Promiscuous RNA Binding Ensures Effective Encapsidation of APOBEC3 Proteins by HIV-1
Article Snippet: Cells and viruses were then resuspended in 1 ml of lysis buffer (50 mM Tris-HCL, pH 7.4; 100 mM NaCl; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and protease inhibitor) and sonicated. .. The RNAs were dephosphorylated using Shrimp alkaline phosphatase (Promega) and a pre-adenylated linker was ligated to the 3’-end of RNAs on beads.

DNA Extraction:

Article Title: Association of LOX-1 gene polymorphisms with cerebral infarction in northern Chinese Han population
Article Snippet: Paragraph title: DNA Extraction and Genotyping ... The PCR cycling program was set at 95°C for 2 min, followed by 11 cycles of 94°C for 20 s, 65°C (decreased 0.5°C per cycle) for 40 s, 72°C for 1.5 min, and then 24 cycles of 94°C for 20 s, 59°C for 30 s, and 72°C for 1.5 min, and a final extension at 72°C for 2 min. Then shrimp alkaline phosphatase (Promega, USA) and Exonuclease I (Epicenter, USA) were added into the PCR products for purification.

Article Title: Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species
Article Snippet: Paragraph title: 3.3. DNA Extraction, PCR Primers, Cycling Profiles and DNA Sequencing ... For sequence analysis PCR-amplified DNA fragments were purified with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following program: 30 min at 37 °C and 15 min at 80 °C.

Nucleic Acid Electrophoresis:

Article Title: Promiscuous RNA Binding Ensures Effective Encapsidation of APOBEC3 Proteins by HIV-1
Article Snippet: The RNAs were dephosphorylated using Shrimp alkaline phosphatase (Promega) and a pre-adenylated linker was ligated to the 3’-end of RNAs on beads. .. RNAs were radiolabeled with P32 -ϒ-ATP and separated using SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose and visualised on film.

Methylation:

Article Title: Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which includes the loss of tumor suppressor genes involved in the control of cell growth and death
Article Snippet: Primers were designed to amplify a product, from bisulphite modified DNA, covering the selected methylation loci (Online Resource 1). .. This was followed by a final extension step at 72°C for 10 min. PCR products were purified by incubation with 0.3 U shrimp alkaline phosphatase (Promega, Madison, WI, USA) and 1.5 U exonuclease I (NEB, Ipswich, MA, USA) at 37°C for 8 min, followed by 15 min at 72°C.

Multiple Displacement Amplification:

Article Title: Non-Invasive Imaging of Tumors by Monitoring Autotaxin Activity Using an Enzyme-Activated Near-Infrared Fluorogenic Substrate
Article Snippet: Shrimp alkaline phosphatase was purchased from Promega (Madison, WI). .. COS-7, MDA-MB-231, and MDA-MB-435 cell lines were purchased from ATCC (Manassas, VA).

Isolation:

Article Title: Promiscuous RNA Binding Ensures Effective Encapsidation of APOBEC3 Proteins by HIV-1
Article Snippet: The RNAs were dephosphorylated using Shrimp alkaline phosphatase (Promega) and a pre-adenylated linker was ligated to the 3’-end of RNAs on beads. .. Pieces of the membrane containing the RNAs of interest were excised and resuspended in PK buffer (100 mM Tris-HCl pH 7.5, 50 mM NaCl, 10 mM EDTA) containing 2 mg/ml proteinase K. RNAs were isolated with phenol/chloroform (Ambion) and precipitated with 2.5 volumes of 100% ethanol, 0.1 volumes of sodium acetate (3 M, pH 5.5) and 0.5 μl of glycoblue.

Subcloning:

Article Title: The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines
Article Snippet: Paragraph title: Sub-cloning into pET-28a (+) vector ... The linearized plasmid was treated with 2 units of shrimp alkaline phosphatase (Promega, Madison, USA) at 37°C for 30 min.

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: Paragraph title: M. tuberculosis cosmid library construction and subcloning analysis of TNF-downregulating hits. ... The plasmid pYUB412 was linearized with XbaI, dephosphorylated with shrimp alkaline phosphatase (Promega), and then digested with BclI to obtain 3,789-bp and 4,773-bp fragments.

Size-exclusion Chromatography:

Article Title: Targeted Deep Sequencing Identifies Rare ‘loss-of-function’ Variants in IFNGR1 for Risk of Atopic Dermatitis Complicated by Eczema Herpeticum
Article Snippet: .. The reaction was then cycled with the following conditions: initial denaturation at 94°C for 12 min; 40 cycles at 94°C for 20 sec, 54°C for 20 sec, and 72°C for 20 sec; final extension at 72°C for 10 min. PCR products were purified using Exonuclease III and Shrimp Alkaline Phosphatase (Promega, Madison, WI). .. Automated sequencing was performed by capillary electrophoresis on an ABI3700 (Applied Biosystems) at the Johns Hopkins Genetic Resources Core Facility and all variants were manually called by visual inspection of the electropherogram, using Sequencher software (Gene Codes Corp., Inc.).

Labeling:

Article Title: Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species
Article Snippet: For sequence analysis PCR-amplified DNA fragments were purified with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following program: 30 min at 37 °C and 15 min at 80 °C. .. Both strands were labeled using a BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to Błaszczyk et al. [ ], and precipitated with ethanol.

Purification:

Article Title: The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines
Article Snippet: The released insert was purified from the agarose gel using the QIAquick Gel Extraction Kit (Cat. # 28704, QIAGEN) and sub-cloned into the pET-28a (+) expression vector. .. The linearized plasmid was treated with 2 units of shrimp alkaline phosphatase (Promega, Madison, USA) at 37°C for 30 min.

Article Title: Association of LOX-1 gene polymorphisms with cerebral infarction in northern Chinese Han population
Article Snippet: .. The PCR cycling program was set at 95°C for 2 min, followed by 11 cycles of 94°C for 20 s, 65°C (decreased 0.5°C per cycle) for 40 s, 72°C for 1.5 min, and then 24 cycles of 94°C for 20 s, 59°C for 30 s, and 72°C for 1.5 min, and a final extension at 72°C for 2 min. Then shrimp alkaline phosphatase (Promega, USA) and Exonuclease I (Epicenter, USA) were added into the PCR products for purification. ..

Article Title: Association of NLRP1 and NLRP3 Polymorphisms with Psoriasis Vulgaris Risk in the Chinese Han Population
Article Snippet: .. The products were purified via digestion of 5 U shrimp alkaline phosphatase (Promega) and 2 U exonuclease I at 37°C for 1 h, followed by 75°C for 15 min. .. The ligation reaction was carried out in 10 μ L of a mixture comprising 1 μ L 10x ligation buffer, 0.25 μ L 80 U/μ L Taq DNA ligase (New England Biolabs), 0.4 μ L 5′ ligation primer mixture (1 μ M), 2 μ L purified PCR products, and 6 μ L ddH2O.

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: For the RNAi vector containing sequences for both ppt-1 and ath-1 , vectors containing ppt-1 and ath-1 individually were purified from overnight bacterial cultures. .. 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C.

Article Title: Non-Invasive Imaging of Tumors by Monitoring Autotaxin Activity Using an Enzyme-Activated Near-Infrared Fluorogenic Substrate
Article Snippet: Reagents Purified human ATX, INPP4A, LPLA2, PF-8380, S32826, BrP-LPA and FS-3 were obtained from Echelon Biosciences, Inc. (Salt Lake City, UT). .. Shrimp alkaline phosphatase was purchased from Promega (Madison, WI).

Article Title: Targeted Deep Sequencing Identifies Rare ‘loss-of-function’ Variants in IFNGR1 for Risk of Atopic Dermatitis Complicated by Eczema Herpeticum
Article Snippet: .. The reaction was then cycled with the following conditions: initial denaturation at 94°C for 12 min; 40 cycles at 94°C for 20 sec, 54°C for 20 sec, and 72°C for 20 sec; final extension at 72°C for 10 min. PCR products were purified using Exonuclease III and Shrimp Alkaline Phosphatase (Promega, Madison, WI). .. Automated sequencing was performed by capillary electrophoresis on an ABI3700 (Applied Biosystems) at the Johns Hopkins Genetic Resources Core Facility and all variants were manually called by visual inspection of the electropherogram, using Sequencher software (Gene Codes Corp., Inc.).

Article Title: Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation
Article Snippet: .. Digestion products were purified by gel extraction (Qiagen) and vectors were treated with shrimp alkaline phosphatase (Promega) to prevent self-ligation. .. Inserts and vectors were ligated using T4 DNA Ligase (NEB) at a 1:1 ratio.

Article Title: Supratentorial and spinal pediatric ependymomas display a hypermethylated phenotype which includes the loss of tumor suppressor genes involved in the control of cell growth and death
Article Snippet: .. This was followed by a final extension step at 72°C for 10 min. PCR products were purified by incubation with 0.3 U shrimp alkaline phosphatase (Promega, Madison, WI, USA) and 1.5 U exonuclease I (NEB, Ipswich, MA, USA) at 37°C for 8 min, followed by 15 min at 72°C. ..

Article Title: A Functional Polymorphism in the Promoter Region of MicroRNA-146a Is Associated with the Risk of Alzheimer Disease and the Rate of Cognitive Decline in Patients
Article Snippet: .. Extension products were purified by a 15-minute incubation with 1U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C and a subsequent 15-minute incubation at 80°C to inactivate the enzyme. .. The purified products (0.5 µl) were mixed with 9 µl of formamide and 0.5 µl of GeneScan-120 LIZ Size Standard (Applied Biosystems) and separated by capillary electrophoresis (ABI PRISM310 Genetic Analyzer; Applied Biosystems).

Article Title: Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species
Article Snippet: .. For sequence analysis PCR-amplified DNA fragments were purified with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following program: 30 min at 37 °C and 15 min at 80 °C. .. Both strands were labeled using a BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to Błaszczyk et al. [ ], and precipitated with ethanol.

Sequencing:

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: 20 μl DNA was digested at 37°C in a reaction volume of 25 μl: pG-L4440-ppt-1 was linearised using Acc65I ; pG-L4440-ath-1 was digested with BsrGI to obtain the ath-1 sequence but retaining complementary 5’ overhangs to enable its ligation into the pG-L4440-ppt-1 vector. .. 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C.

Article Title: Targeted Deep Sequencing Identifies Rare ‘loss-of-function’ Variants in IFNGR1 for Risk of Atopic Dermatitis Complicated by Eczema Herpeticum
Article Snippet: Two variants (rs11914 and rs10457655), along with the three rare missense variants (Val14Met, Val61Ile and Tyr397Cys) were validated using TaqMan Allelic Discrimination Assays on the 7900HT Sequence Detection System (Applied Biosystems), as previously described ) with default parameters (forward primer sequence: 5′-ACCGTTTAGGCGTCTATGGAG-3′, reverse primer sequence: 5′-CCCATTGCTCTCCTTGGGAAT-3′). .. The reaction was then cycled with the following conditions: initial denaturation at 94°C for 12 min; 40 cycles at 94°C for 20 sec, 54°C for 20 sec, and 72°C for 20 sec; final extension at 72°C for 10 min. PCR products were purified using Exonuclease III and Shrimp Alkaline Phosphatase (Promega, Madison, WI).

Article Title: Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation
Article Snippet: Heavy and light chain DNA of mouse anti-mouse CD20 monoclonal antibody 5D2.16.8 (previously cloned from a hybridoma cell line into mouse IgG2b and mouse kappa) was diluted to less than 500 ng/µl and amplified through PCR with a premix Taq polymerase kit (Takara) using forward primers corresponding to antibody framework region and human vector signal sequence and reverse primers corresponding to antibody framework region and human IgG4 or kappa CH1 (primers shown below). .. Digestion products were purified by gel extraction (Qiagen) and vectors were treated with shrimp alkaline phosphatase (Promega) to prevent self-ligation.

Article Title: Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species
Article Snippet: .. For sequence analysis PCR-amplified DNA fragments were purified with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following program: 30 min at 37 °C and 15 min at 80 °C. .. Both strands were labeled using a BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to Błaszczyk et al. [ ], and precipitated with ethanol.

Gel Extraction:

Article Title: The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines
Article Snippet: The released insert was purified from the agarose gel using the QIAquick Gel Extraction Kit (Cat. # 28704, QIAGEN) and sub-cloned into the pET-28a (+) expression vector. .. The linearized plasmid was treated with 2 units of shrimp alkaline phosphatase (Promega, Madison, USA) at 37°C for 30 min.

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C. .. The products were subjected to agarose gel electrophoresis and the pG-L4440-ppt-1 and ath-1 bands were excised and purified into 50 μl using a GenElute™ Gel Extraction Kit (Sigma, Dorset, UK).

Article Title: Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation
Article Snippet: .. Digestion products were purified by gel extraction (Qiagen) and vectors were treated with shrimp alkaline phosphatase (Promega) to prevent self-ligation. .. Inserts and vectors were ligated using T4 DNA Ligase (NEB) at a 1:1 ratio.

Plasmid Preparation:

Article Title: The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines
Article Snippet: .. The linearized plasmid was treated with 2 units of shrimp alkaline phosphatase (Promega, Madison, USA) at 37°C for 30 min. .. The ligation reaction was carried out in a tube containing 2 μL (50 ng) of pET28a (+), 2 μL (100 ng) of IFNε cDNA insert, 1 μL 10X ligase buffer, and 1 μL (2 units) of ligase enzyme.

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: .. The plasmid pYUB412 was linearized with XbaI, dephosphorylated with shrimp alkaline phosphatase (Promega), and then digested with BclI to obtain 3,789-bp and 4,773-bp fragments. .. M. tuberculosis H37Rv genomic DNA was prepared from a 20-ml culture (OD600 , ~3) grown in Middlebrook 7H9 supplemented with 10% OADC, 0.2% glycerol, and 0.05% Tween 80 as previously described , with the following modification: the genomic DNA, once precipitated in isopropanol, was spooled and immersed in ethanol and was then air-dried instead of being spun down.

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: 20 μl DNA was digested at 37°C in a reaction volume of 25 μl: pG-L4440-ppt-1 was linearised using Acc65I ; pG-L4440-ath-1 was digested with BsrGI to obtain the ath-1 sequence but retaining complementary 5’ overhangs to enable its ligation into the pG-L4440-ppt-1 vector. .. 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C.

Article Title: Increased in vivo effector function of human IgG4 isotype antibodies through afucosylation
Article Snippet: The light chain PCR product and pRK human kappa LPG3 vector were digested using KpnI and EcoRI restriction enzymes (NEB) according to manufacturer recommendations. .. Digestion products were purified by gel extraction (Qiagen) and vectors were treated with shrimp alkaline phosphatase (Promega) to prevent self-ligation.

Software:

Article Title: Association of NLRP1 and NLRP3 Polymorphisms with Psoriasis Vulgaris Risk in the Chinese Han Population
Article Snippet: The products were purified via digestion of 5 U shrimp alkaline phosphatase (Promega) and 2 U exonuclease I at 37°C for 1 h, followed by 75°C for 15 min. .. The raw data were analyzed using GeneMapper 4.1 software (Applied Biosystems, Foster City, CA, USA).

Article Title: Targeted Deep Sequencing Identifies Rare ‘loss-of-function’ Variants in IFNGR1 for Risk of Atopic Dermatitis Complicated by Eczema Herpeticum
Article Snippet: The reaction was then cycled with the following conditions: initial denaturation at 94°C for 12 min; 40 cycles at 94°C for 20 sec, 54°C for 20 sec, and 72°C for 20 sec; final extension at 72°C for 10 min. PCR products were purified using Exonuclease III and Shrimp Alkaline Phosphatase (Promega, Madison, WI). .. Automated sequencing was performed by capillary electrophoresis on an ABI3700 (Applied Biosystems) at the Johns Hopkins Genetic Resources Core Facility and all variants were manually called by visual inspection of the electropherogram, using Sequencher software (Gene Codes Corp., Inc.).

Article Title: A Functional Polymorphism in the Promoter Region of MicroRNA-146a Is Associated with the Risk of Alzheimer Disease and the Rate of Cognitive Decline in Patients
Article Snippet: Extension products were purified by a 15-minute incubation with 1U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C and a subsequent 15-minute incubation at 80°C to inactivate the enzyme. .. The results were analyzed with GeneMapper 3.0 software (Applied Biosystems).

Multiplex Assay:

Article Title: Association of NLRP1 and NLRP3 Polymorphisms with Psoriasis Vulgaris Risk in the Chinese Han Population
Article Snippet: Multiplex polymerase chain reaction (PCR) was used to amplify fragments including these eight SNP sites (see Tables and for the primer information). .. The products were purified via digestion of 5 U shrimp alkaline phosphatase (Promega) and 2 U exonuclease I at 37°C for 1 h, followed by 75°C for 15 min.

Article Title: A Functional Polymorphism in the Promoter Region of MicroRNA-146a Is Associated with the Risk of Alzheimer Disease and the Rate of Cognitive Decline in Patients
Article Snippet: Briefly, the SNapShot reaction was carried out in a 10 µl final volume containing SNapShot Multiplex Ready Mix (5 µl), primer mix (0.02–0.6 µmol/L), and templates (4 µl) consisting of the multiplex polymerase chain reaction (PCR) products, which had been purified with the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). .. Extension products were purified by a 15-minute incubation with 1U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C and a subsequent 15-minute incubation at 80°C to inactivate the enzyme.

Positron Emission Tomography:

Article Title: The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines
Article Snippet: Paragraph title: Sub-cloning into pET-28a (+) vector ... The linearized plasmid was treated with 2 units of shrimp alkaline phosphatase (Promega, Madison, USA) at 37°C for 30 min.

Agarose Gel Electrophoresis:

Article Title: The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines
Article Snippet: The released insert was purified from the agarose gel using the QIAquick Gel Extraction Kit (Cat. # 28704, QIAGEN) and sub-cloned into the pET-28a (+) expression vector. .. The linearized plasmid was treated with 2 units of shrimp alkaline phosphatase (Promega, Madison, USA) at 37°C for 30 min.

Article Title: A systematic analysis of protein palmitoylation in Caenorhabditis elegans
Article Snippet: 5’-phosphates were removed from linearised pG-L4440-ppt-1 by incubating with 2 μl shrimp alkaline phosphatase (Promega, Madison, WI, USA) for 15 minutes at 37°C. .. The products were subjected to agarose gel electrophoresis and the pG-L4440-ppt-1 and ath-1 bands were excised and purified into 50 μl using a GenElute™ Gel Extraction Kit (Sigma, Dorset, UK).

In Vitro:

Article Title: The role of the priming loop in Influenza A virus RNA synthesis
Article Snippet: Paragraph title: In vitro dinucleotide synthesis assays ... The reactions were incubated at 30 °C for 2 h and depending on the experiment then either i) heated to 95 °C for 2 min, treated with 0.5 U shrimp alkaline phosphatase (SAP, Promega) for 30 min at 37 °C, and subsequently stopped with 3 μl stopping solution (90% formamide, 10 mM EDTA, 0.01% bromophenol blue and 0.01% xylene cyanol), or ii) directly stopped with 3 μl stopping solution.

Spectrophotometry:

Article Title: Association of LOX-1 gene polymorphisms with cerebral infarction in northern Chinese Han population
Article Snippet: DNA purity was assessed by spectrophotometry, and DNA samples were stored at -20°C. .. The PCR cycling program was set at 95°C for 2 min, followed by 11 cycles of 94°C for 20 s, 65°C (decreased 0.5°C per cycle) for 40 s, 72°C for 1.5 min, and then 24 cycles of 94°C for 20 s, 59°C for 30 s, and 72°C for 1.5 min, and a final extension at 72°C for 2 min. Then shrimp alkaline phosphatase (Promega, USA) and Exonuclease I (Epicenter, USA) were added into the PCR products for purification.

Immunoprecipitation:

Article Title: Promiscuous RNA Binding Ensures Effective Encapsidation of APOBEC3 Proteins by HIV-1
Article Snippet: Paragraph title: Individual-nucleotide resolution UV cross-linking and immunoprecipitation ... The RNAs were dephosphorylated using Shrimp alkaline phosphatase (Promega) and a pre-adenylated linker was ligated to the 3’-end of RNAs on beads.

DNA Purification:

Article Title: Association of LOX-1 gene polymorphisms with cerebral infarction in northern Chinese Han population
Article Snippet: DNA Extraction and Genotyping Genomic DNA was extracted from EDTA-anticoagulated peripheral blood by a DNA Purification Kit (Promega, Madison, USA). .. The PCR cycling program was set at 95°C for 2 min, followed by 11 cycles of 94°C for 20 s, 65°C (decreased 0.5°C per cycle) for 40 s, 72°C for 1.5 min, and then 24 cycles of 94°C for 20 s, 59°C for 30 s, and 72°C for 1.5 min, and a final extension at 72°C for 2 min. Then shrimp alkaline phosphatase (Promega, USA) and Exonuclease I (Epicenter, USA) were added into the PCR products for purification.

FLAG-tag:

Article Title: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: .. DNA plasmids and Antibodies Mammalian expression vectors pCIG2 (modified to comprise an N-terminal FLAG tag) and pcDNA3 (modified to comprise an N-terminal FLAG or HA epitope tag) were digested with EcoRI and treated with shrimp alkaline phosphatase (Promega, Australia) before ligation to mouse Eurl cDNA amplified by PCR using primers which encoded flanking MfeI restriction sites, together with a template (IMAGE:3966397 also known as D16Ertd472e, Genbank Accession Number BC019957). ..

Lysis:

Article Title: Promiscuous RNA Binding Ensures Effective Encapsidation of APOBEC3 Proteins by HIV-1
Article Snippet: Cells and viruses were then resuspended in 1 ml of lysis buffer (50 mM Tris-HCL, pH 7.4; 100 mM NaCl; 1% NP-40; 0.1% SDS; 0.5% sodium deoxycholate and protease inhibitor) and sonicated. .. The RNAs were dephosphorylated using Shrimp alkaline phosphatase (Promega) and a pre-adenylated linker was ligated to the 3’-end of RNAs on beads.

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    Promega tsap thermosensitive alkaline phosphatase
    Tsap Thermosensitive Alkaline Phosphatase, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
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    ΔNp63α overexpression results in increased nuclear levels of transcriptionally active phosphorylated c-Rel in murine keratinocytes A) Elevated Δ Np63α expression specifically enhances nuclear levels of the NF-κ B subunit c-Rel. Western blots of nuclear extracts from primary murine keratinocytes harvested 21h post-adenoviral introduction of human ΔNp63α or β-gal. Cultures were maintained in medium containing 0.05mM Ca 2+ throughout or exposed to 0.12 mM Ca 2+ for the final 4h. C-Rel levels are increased in the nuclei of ΔNp63α - overexpressing keratinocytes. B) c-Rel is phosphorylated in response to elevated ΔNp63α. Western analysis of nuclear extracts from keratinocytes overexpressing ΔNp63α or β-gal cultured under control conditions (top) or in the presence of the thiol modifier N-ethylmaleimide (NEM) at concentrations noted for 21h following adenoviral introduction to block in vivo phosphorylation (bottom left). The upper species is eliminated in the presence of NEM. Phosphorylation was confirmed by western analysis of nuclear extracts derived from keratinocytes overexpressing ΔNp63α incubated in the presence or absence of Shrimp Alkaline Phosphatase <t>(SAP)</t> for <t>3h</t> at 37°C (bottom right). A non-incubated control nuclear extract (NE) is included for visual reference. SAP treatment results in the loss of the upper phosphorylated species. C) ΔNp63α overexpression results in NF-κ B-mediated transactivation; α-domain of ΔNp63α required. NF-κB responsive reporter gene activity in keratinocytes overexpressing ΔNp63α, ΔNp63 p40 (ΔNp63 lacking α-domain) or β-gal. Samples were harvested 24h post-transfection. Results depict mean +/− s.d. of triplicate samples of a representative experiment performed thrice. D) c-Rel is required for NF-κ B-mediated transactivation following ΔNp63α overexpression. Keratinocytes were co-transfected with NF-κB reporter construct and c-Rel-targeting siRNA or control siRNA, 24h prior to adenoviral infection with ΔNp63α or β-gal. Samples were harvested 24h post adenoviral introduction. Right side: Western blot reveals depletion of c-Rel in whole cell lysates at time of harvest (NT: non targeting siRNA). Left side: Fold increase in NF-κB reporter gene activity in ΔNp63α overexpressing cultures compared to β-gal control cultures in the presence or absence of c-Rel siRNA. Triplicate wells were averaged and are presented as fold increase relative to β-gal controls that are normalized to 1.0. Experiment was performed twice with consistent results; representative experiment shown.
    Shrimp Alkaline Phosphatase Sap, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega shrimp alkaline phosphatase sirnas
    Immunostimulatory properties of <t>T7-siRNAs</t> and c-siRNAs in chicken cells. IFN-β mRNA levels were measured in DF-1 cells transfected with T7-siRNAs (white bars, 20 pmol) or c-siRNAs (black bars, 20 pmol) at (A) 8 h or (B) 24 h. a = p
    Shrimp Alkaline Phosphatase Sirnas, supplied by Promega, used in various techniques. Bioz Stars score: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ΔNp63α overexpression results in increased nuclear levels of transcriptionally active phosphorylated c-Rel in murine keratinocytes A) Elevated Δ Np63α expression specifically enhances nuclear levels of the NF-κ B subunit c-Rel. Western blots of nuclear extracts from primary murine keratinocytes harvested 21h post-adenoviral introduction of human ΔNp63α or β-gal. Cultures were maintained in medium containing 0.05mM Ca 2+ throughout or exposed to 0.12 mM Ca 2+ for the final 4h. C-Rel levels are increased in the nuclei of ΔNp63α - overexpressing keratinocytes. B) c-Rel is phosphorylated in response to elevated ΔNp63α. Western analysis of nuclear extracts from keratinocytes overexpressing ΔNp63α or β-gal cultured under control conditions (top) or in the presence of the thiol modifier N-ethylmaleimide (NEM) at concentrations noted for 21h following adenoviral introduction to block in vivo phosphorylation (bottom left). The upper species is eliminated in the presence of NEM. Phosphorylation was confirmed by western analysis of nuclear extracts derived from keratinocytes overexpressing ΔNp63α incubated in the presence or absence of Shrimp Alkaline Phosphatase (SAP) for 3h at 37°C (bottom right). A non-incubated control nuclear extract (NE) is included for visual reference. SAP treatment results in the loss of the upper phosphorylated species. C) ΔNp63α overexpression results in NF-κ B-mediated transactivation; α-domain of ΔNp63α required. NF-κB responsive reporter gene activity in keratinocytes overexpressing ΔNp63α, ΔNp63 p40 (ΔNp63 lacking α-domain) or β-gal. Samples were harvested 24h post-transfection. Results depict mean +/− s.d. of triplicate samples of a representative experiment performed thrice. D) c-Rel is required for NF-κ B-mediated transactivation following ΔNp63α overexpression. Keratinocytes were co-transfected with NF-κB reporter construct and c-Rel-targeting siRNA or control siRNA, 24h prior to adenoviral infection with ΔNp63α or β-gal. Samples were harvested 24h post adenoviral introduction. Right side: Western blot reveals depletion of c-Rel in whole cell lysates at time of harvest (NT: non targeting siRNA). Left side: Fold increase in NF-κB reporter gene activity in ΔNp63α overexpressing cultures compared to β-gal control cultures in the presence or absence of c-Rel siRNA. Triplicate wells were averaged and are presented as fold increase relative to β-gal controls that are normalized to 1.0. Experiment was performed twice with consistent results; representative experiment shown.

    Journal: Cancer research

    Article Title: The p53 homologue ?Np63? interacts with the NF-?B pathway to modulate epithelial cell growth

    doi: 10.1158/0008-5472.CAN-07-6123

    Figure Lengend Snippet: ΔNp63α overexpression results in increased nuclear levels of transcriptionally active phosphorylated c-Rel in murine keratinocytes A) Elevated Δ Np63α expression specifically enhances nuclear levels of the NF-κ B subunit c-Rel. Western blots of nuclear extracts from primary murine keratinocytes harvested 21h post-adenoviral introduction of human ΔNp63α or β-gal. Cultures were maintained in medium containing 0.05mM Ca 2+ throughout or exposed to 0.12 mM Ca 2+ for the final 4h. C-Rel levels are increased in the nuclei of ΔNp63α - overexpressing keratinocytes. B) c-Rel is phosphorylated in response to elevated ΔNp63α. Western analysis of nuclear extracts from keratinocytes overexpressing ΔNp63α or β-gal cultured under control conditions (top) or in the presence of the thiol modifier N-ethylmaleimide (NEM) at concentrations noted for 21h following adenoviral introduction to block in vivo phosphorylation (bottom left). The upper species is eliminated in the presence of NEM. Phosphorylation was confirmed by western analysis of nuclear extracts derived from keratinocytes overexpressing ΔNp63α incubated in the presence or absence of Shrimp Alkaline Phosphatase (SAP) for 3h at 37°C (bottom right). A non-incubated control nuclear extract (NE) is included for visual reference. SAP treatment results in the loss of the upper phosphorylated species. C) ΔNp63α overexpression results in NF-κ B-mediated transactivation; α-domain of ΔNp63α required. NF-κB responsive reporter gene activity in keratinocytes overexpressing ΔNp63α, ΔNp63 p40 (ΔNp63 lacking α-domain) or β-gal. Samples were harvested 24h post-transfection. Results depict mean +/− s.d. of triplicate samples of a representative experiment performed thrice. D) c-Rel is required for NF-κ B-mediated transactivation following ΔNp63α overexpression. Keratinocytes were co-transfected with NF-κB reporter construct and c-Rel-targeting siRNA or control siRNA, 24h prior to adenoviral infection with ΔNp63α or β-gal. Samples were harvested 24h post adenoviral introduction. Right side: Western blot reveals depletion of c-Rel in whole cell lysates at time of harvest (NT: non targeting siRNA). Left side: Fold increase in NF-κB reporter gene activity in ΔNp63α overexpressing cultures compared to β-gal control cultures in the presence or absence of c-Rel siRNA. Triplicate wells were averaged and are presented as fold increase relative to β-gal controls that are normalized to 1.0. Experiment was performed twice with consistent results; representative experiment shown.

    Article Snippet: Nuclear extracts were incubated in 1X SAP buffer +/− 10U Shrimp Alkaline Phosphatase (SAP) (Promega, Madison, WI) for 3h at 37°C followed by inactivation at 65°C.

    Techniques: Over Expression, Expressing, Western Blot, Cell Culture, Blocking Assay, In Vivo, Derivative Assay, Incubation, Activity Assay, Transfection, Construct, Infection

    Immunostimulatory properties of T7-siRNAs and c-siRNAs in chicken cells. IFN-β mRNA levels were measured in DF-1 cells transfected with T7-siRNAs (white bars, 20 pmol) or c-siRNAs (black bars, 20 pmol) at (A) 8 h or (B) 24 h. a = p

    Journal: PLoS ONE

    Article Title: Immunostimulatory Motifs Enhance Antiviral siRNAs Targeting Highly Pathogenic Avian Influenza H5N1

    doi: 10.1371/journal.pone.0021552

    Figure Lengend Snippet: Immunostimulatory properties of T7-siRNAs and c-siRNAs in chicken cells. IFN-β mRNA levels were measured in DF-1 cells transfected with T7-siRNAs (white bars, 20 pmol) or c-siRNAs (black bars, 20 pmol) at (A) 8 h or (B) 24 h. a = p

    Article Snippet: Treatment of siRNAs with shrimp alkaline phosphatase siRNAs synthesized using T7 RNA polymerase were treated with shrimp alkaline phosphatase (SAP) according to manufacturers guidelines (Promega).

    Techniques: Transfection