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Promega shrimp alkaline phosphatase
Shrimp Alkaline Phosphatase, supplied by Promega, used in various techniques. Bioz Stars score: 75/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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shrimp alkaline phosphatase - by Bioz Stars, 2019-12
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Clone Assay:

Article Title: Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA
Article Snippet: Paragraph title: Cloning and preparation of human U1- and U2-snRNA by in vitro transcription ... In order to eliminate 5′-triphosphate moieties from in vitro transcribed U1-snRNA that may artificially activate RIG-I , the molecule was treated with shrimp alkaline phosphatase (SAP; Promega, Mannheim, Germany).

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: Since patients diagnosed with Down Syndrome show clear evidence of abnormal neuronal development, including reduced brain volumes , disorganised cortical layers indicative of aberrant neuronal positioning , and reduced dendritic spine densities on cortical neurons ; we suggest that altered EURL dosage contributes to such neurological deficits in Down Syndrome. .. 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). .. A similar strategy was employed to clone mouse Ccdc85b cDNA from IMAGE:5710206 (Genbank Accession Number BC058411).

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: FGF-2 Gene Polymorphism in Osteoporosis among Guangxi’s Zhuang Chinese
Article Snippet: The gene amplification was carried out in PCR reactions of 20 μL, which included 2 μL of 10×buffer, 2.2 μL of MgCl2 (25 mmol/L), 0.8 μL of dNTPs (10 mmol/L), 1 μL for each upstream and downstream primers (10 μmol/L), 1 μL of template, 0.2 μL of Taq DNA polymerase, and an appropriate amount of double-distilled water to obtain 20 μL volume. .. PCR reaction conditions were as follows: 95 °C for 2 min; 11 cycles of 94 °C for 20 s, 65–0.5 °C/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; 72 °C for 2 min; and 4 °C.PCR products were firstly purified with shrimp alkaline phosphatase (SAP, Promega, Madison, WI, USA) and Exonuclease I (EXO I, Epicentre, Wisconsin, USA) and then were used for the extension reaction with SNaPshot Multiplex kit (Applied Biosystems, Foster, CA, USA).

Article Title: Functional Variants in NFKBIE and RTKN2 Involved in Activation of the NF-?B Pathway Are Associated with Rheumatoid Arthritis in Japanese
Article Snippet: DNA fragments were amplified with the appropriate primers ( ). .. Purification of PCR products was performed with Exonuclease I (New England Biolabs, Ipswich, MA, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA).

Synthesized:

Article Title: FGF-2 Gene Polymorphism in Osteoporosis among Guangxi’s Zhuang Chinese
Article Snippet: Primer 5 software was used to design primers, which were synthesized by GENESKY (Shanghai Genesky Biotech Co., Ltd., Shanghai, China). .. PCR reaction conditions were as follows: 95 °C for 2 min; 11 cycles of 94 °C for 20 s, 65–0.5 °C/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; 72 °C for 2 min; and 4 °C.PCR products were firstly purified with shrimp alkaline phosphatase (SAP, Promega, Madison, WI, USA) and Exonuclease I (EXO I, Epicentre, Wisconsin, USA) and then were used for the extension reaction with SNaPshot Multiplex kit (Applied Biosystems, Foster, CA, USA).

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: 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). .. 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).

Electrophoresis:

Article Title: Phylogeography of Eastern Grey Kangaroos, Macropus giganteus, Suggests a Mesic Refugium in Eastern Australia
Article Snippet: PCR products were purified by adding 5 U exonuclease I, Escherichia coli (Fermentas, Scoresby) and 0.5 U shrimp alkaline phosphatase (Promega, Hawthorn) and incubating at 37°C for 30 minutes followed by an enzyme inactivation step of 80°C for 15 minutes. .. PCR products were purified by adding 5 U exonuclease I, Escherichia coli (Fermentas, Scoresby) and 0.5 U shrimp alkaline phosphatase (Promega, Hawthorn) and incubating at 37°C for 30 minutes followed by an enzyme inactivation step of 80°C for 15 minutes.

Incubation:

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 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.

Article Title: Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA
Article Snippet: In order to eliminate 5′-triphosphate moieties from in vitro transcribed U1-snRNA that may artificially activate RIG-I , the molecule was treated with shrimp alkaline phosphatase (SAP; Promega, Mannheim, Germany). .. For that purpose, a protocol proven to effectively remove radiolabeled 5′-phosphate ends from in vitro transcribed U1-snRNA by at least 97% was used (data not shown).

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 conditions were; 95°C for 5 min followed by 40 cycles of 95°C for 30 s, an annealing temperature specific to each primer pair (Online Resource 1) for 1 min and 72°C for 1 min. .. 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. .. Sequencing reactions were undertaken by Source Bioscience (Nottingham, UK).

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: The cycling program included 25 cycles of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 40 seconds. .. 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: The role of the priming loop in Influenza A virus RNA synthesis
Article Snippet: Briefly, for pppApG synthesis assay we incubated a reaction mixture containing 0.05 μM [α-32 P]GTP (3000 Ci/mmole, Perking-Elmer), 1 mM ATP, 5 mM MgCl2 , 1 mM DTT, 1 U/μl RNasin (Promega), 0.7 μM of promoter, 5% glycerol, 0.05% NP-40, 75 mM NaCl, 10 mM Hepes pH 7.5, and 5 ng/μl RdRp. .. 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. .. The level of [α-32 P]GMP incorporation was visualised using phosphorimaging as described above.

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: Contribution of DNA repair xeroderma pigmentosum group D genotypes to pancreatic cancer risk in the Chinese Han population
Article Snippet: The PCR products were labeled by the alkaline phosphatase-labeled streptavidin biotin/exonuclease I (Exo I) method, using 1 U of shrimp alkaline phosphatase (SAP; Promega, Madison, WI, USA) and 1 U of Exo I (New England Biolabs, Beverly, MA, USA). .. The PCR cycling conditions of the primer extension reaction were as follows: pre-denaturation at 96 °C for 1 min, followed by 28 cycles of 96 °C for 10 s, 50 °C for 5 s and 60 °C for 30 s, followed by an extension step at 60 °C for 1 min, and cooling to 4 °C.

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.

Article Title: Renal Function and Klotho Gene Polymorphisms among Uygur and Kazak Populations in Xinjiang, China
Article Snippet: Briefly, the SNaPshot reaction was carried out in a 10 μL final volume containing SNaPshot Multiplex Ready Mix (5 μL), primer mix (1 μmol/L), and templates (2 μL). .. The cycling program condition was: denaturation at 96°C for 1 min, followed by 28 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. The extension products were purified by incubation with 1 U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C for 60 min. .. 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 then separated by capillary electrophoresis (ABI PRISM3130 Genetic Analyzer; Applied Biosystems).

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. .. 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.

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: Since patients diagnosed with Down Syndrome show clear evidence of abnormal neuronal development, including reduced brain volumes , disorganised cortical layers indicative of aberrant neuronal positioning , and reduced dendritic spine densities on cortical neurons ; we suggest that altered EURL dosage contributes to such neurological deficits in Down Syndrome. .. 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). .. A similar strategy was employed to clone mouse Ccdc85b cDNA from IMAGE:5710206 (Genbank Accession Number BC058411).

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: Since patients diagnosed with Down Syndrome show clear evidence of abnormal neuronal development, including reduced brain volumes , disorganised cortical layers indicative of aberrant neuronal positioning , and reduced dendritic spine densities on cortical neurons ; we suggest that altered EURL dosage contributes to such neurological deficits in Down Syndrome. .. 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). .. A similar strategy was employed to clone mouse Ccdc85b cDNA from IMAGE:5710206 (Genbank Accession Number BC058411).

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.

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.

High Performance Liquid Chromatography:

Article Title: Nucleotide Bias Observed with a Short SELEX RNA Aptamer Library
Article Snippet: The triphosphate groups from the nucleotides (ribonucleotides and deoxynucleotides) were removed using shrimp alkaline phosphatase (Promega, M9910). .. The triphosphate groups from the nucleotides (ribonucleotides and deoxynucleotides) were removed using shrimp alkaline phosphatase (Promega, M9910).

Gas Chromatography:

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.

Chromatography:

Article Title: Nucleotide Bias Observed with a Short SELEX RNA Aptamer Library
Article Snippet: Paragraph title: High-performance liquid chromatography-based quantitation of NTP concentrations ... The triphosphate groups from the nucleotides (ribonucleotides and deoxynucleotides) were removed using shrimp alkaline phosphatase (Promega, M9910).

Ligation:

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 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.

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: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: Since patients diagnosed with Down Syndrome show clear evidence of abnormal neuronal development, including reduced brain volumes , disorganised cortical layers indicative of aberrant neuronal positioning , and reduced dendritic spine densities on cortical neurons ; we suggest that altered EURL dosage contributes to such neurological deficits in Down Syndrome. .. 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). .. A similar strategy was employed to clone mouse Ccdc85b cDNA from IMAGE:5710206 (Genbank Accession Number BC058411).

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.

Hemagglutination Assay:

Article Title: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: Since patients diagnosed with Down Syndrome show clear evidence of abnormal neuronal development, including reduced brain volumes , disorganised cortical layers indicative of aberrant neuronal positioning , and reduced dendritic spine densities on cortical neurons ; we suggest that altered EURL dosage contributes to such neurological deficits in Down Syndrome. .. 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). .. A similar strategy was employed to clone mouse Ccdc85b cDNA from IMAGE:5710206 (Genbank Accession Number BC058411).

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: Diversity of Pea-Associated F. proliferatum and F. verticillioides Populations Revealed by FUM1 Sequence Analysis and Fumonisin Biosynthesis
Article Snippet: Paragraph title: 4.4. DNA Sequencing, Analysis and Phylogeny Reconstruction ... PCR-amplified DNA fragments were purified for sequence analysis with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following program: 30 min at 37 °C, followed by 15 min at 80 °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.

Sequencing:

Article Title: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: 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). .. 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: Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA
Article Snippet: A T7 promoter sequence was introduced directly in front of the U1-snRNA gene. .. In order to eliminate 5′-triphosphate moieties from in vitro transcribed U1-snRNA that may artificially activate RIG-I , the molecule was treated with shrimp alkaline phosphatase (SAP; Promega, Mannheim, Germany).

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).

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: Diversity of Pea-Associated F. proliferatum and F. verticillioides Populations Revealed by FUM1 Sequence Analysis and Fumonisin Biosynthesis
Article Snippet: Amplicons were electrophoresed in 1.5% agarose gels (Invitrogen, Carlsbad, CA, USA) with ethidium bromide. .. PCR-amplified DNA fragments were purified for sequence analysis with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following program: 30 min at 37 °C, followed by 15 min at 80 °C. .. Both strands were labeled using the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to Błaszczyk et al. [ ] and the manufacturer’s instructions.

Article Title: Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample
Article Snippet: The PCR primers were designed with the free internet tool Primer3 v.0.4.0 ( http://bioinfo.ut.ee/primer3-0.4.0/ ). .. PCR products were purified by Exo/SAP digestion with Exonuclease I (New England Biolabs, Beverly, MA) and Shrimp Alkaline Phosphatase (Promega, San Diego, USA) and directly sequenced using the ABI PRISM BigDye® Terminator v1.1 Cycle Sequencing Kit and the ABI 3730 sequencing instrument (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. .. All primer sequences and PCR conditions can be obtained on request from the authors.

Article Title: Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species
Article Snippet: Amplicons were electrophoresed in 1.5% agarose gels (Invitrogen, Carlsbad, CA, USA) with ethidium bromide. .. 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.

Article Title: Phylogeography of Eastern Grey Kangaroos, Macropus giganteus, Suggests a Mesic Refugium in Eastern Australia
Article Snippet: Paragraph title: mtDNA sequencing ... PCR products were purified by adding 5 U exonuclease I, Escherichia coli (Fermentas, Scoresby) and 0.5 U shrimp alkaline phosphatase (Promega, Hawthorn) and incubating at 37°C for 30 minutes followed by an enzyme inactivation step of 80°C for 15 minutes.

Article Title: Functional Variants in NFKBIE and RTKN2 Involved in Activation of the NF-?B Pathway Are Associated with Rheumatoid Arthritis in Japanese
Article Snippet: Unknown variants in the coding sequences of NFKBIE and RTKN2 were revealed by directly sequencing the DNA of 48 individuals affected with RA. .. Purification of PCR products was performed with Exonuclease I (New England Biolabs, Ipswich, MA, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA).

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.

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.

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. .. 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: Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample
Article Snippet: PCR products were purified by Exo/SAP digestion with Exonuclease I (New England Biolabs, Beverly, MA) and Shrimp Alkaline Phosphatase (Promega, San Diego, USA) and directly sequenced using the ABI PRISM BigDye® Terminator v1.1 Cycle Sequencing Kit and the ABI 3730 sequencing instrument (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. .. PCR products were purified by Exo/SAP digestion with Exonuclease I (New England Biolabs, Beverly, MA) and Shrimp Alkaline Phosphatase (Promega, San Diego, USA) and directly sequenced using the ABI PRISM BigDye® Terminator v1.1 Cycle Sequencing Kit and the ABI 3730 sequencing instrument (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions.

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. .. RNAs were radiolabeled with P32 -ϒ-ATP and separated using SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose and visualised on film.

Subcloning:

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.

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.

Article Title: FGF-2 Gene Polymorphism in Osteoporosis among Guangxi’s Zhuang Chinese
Article Snippet: The gene amplification was carried out in PCR reactions of 20 μL, which included 2 μL of 10×buffer, 2.2 μL of MgCl2 (25 mmol/L), 0.8 μL of dNTPs (10 mmol/L), 1 μL for each upstream and downstream primers (10 μmol/L), 1 μL of template, 0.2 μL of Taq DNA polymerase, and an appropriate amount of double-distilled water to obtain 20 μL volume. .. PCR reaction conditions were as follows: 95 °C for 2 min; 11 cycles of 94 °C for 20 s, 65–0.5 °C/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; 72 °C for 2 min; and 4 °C.PCR products were firstly purified with shrimp alkaline phosphatase (SAP, Promega, Madison, WI, USA) and Exonuclease I (EXO I, Epicentre, Wisconsin, USA) and then were used for the extension reaction with SNaPshot Multiplex kit (Applied Biosystems, Foster, CA, USA). .. The extension product after purification by SAP was sampled on ABI3130xl.

Article Title: Renal Function and Klotho Gene Polymorphisms among Uygur and Kazak Populations in Xinjiang, China
Article Snippet: Briefly, the SNaPshot reaction was carried out in a 10 μL final volume containing SNaPshot Multiplex Ready Mix (5 μL), primer mix (1 μmol/L), and templates (2 μL). .. The cycling program condition was: denaturation at 96°C for 1 min, followed by 28 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. The extension products were purified by incubation with 1 U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C for 60 min.

Labeling:

Article Title: Contribution of DNA repair xeroderma pigmentosum group D genotypes to pancreatic cancer risk in the Chinese Han population
Article Snippet: PCR products were purified using an Exo/SAP protocol (Applied Biosystems, Foster City, CA, USA) according to the manufaccturers instructions. .. The PCR products were labeled by the alkaline phosphatase-labeled streptavidin biotin/exonuclease I (Exo I) method, using 1 U of shrimp alkaline phosphatase (SAP; Promega, Madison, WI, USA) and 1 U of Exo I (New England Biolabs, Beverly, MA, USA). .. The SNaPshot extension reaction system consisted of 5 μL of the SNaPshot multiple reaction kit agents (Applied Biosystems), 2 μL of purified multiple PCR products, and 1 μL of the extension primer mixture, and completing the volume to 10 μL with ultra pure water.

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. .. 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: 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 conditions were; 95°C for 5 min followed by 40 cycles of 95°C for 30 s, an annealing temperature specific to each primer pair (Online Resource 1) for 1 min and 72°C for 1 min. .. 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. .. Sequencing reactions were undertaken by Source Bioscience (Nottingham, UK).

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: The cycling program included 25 cycles of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 40 seconds. .. 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: 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. .. The probes for LDR are shown in Table .

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: Contribution of DNA repair xeroderma pigmentosum group D genotypes to pancreatic cancer risk in the Chinese Han population
Article Snippet: PCR products were purified using an Exo/SAP protocol (Applied Biosystems, Foster City, CA, USA) according to the manufaccturers instructions. .. The PCR products were labeled by the alkaline phosphatase-labeled streptavidin biotin/exonuclease I (Exo I) method, using 1 U of shrimp alkaline phosphatase (SAP; Promega, Madison, WI, USA) and 1 U of Exo I (New England Biolabs, Beverly, MA, USA).

Article Title: FGF-2 Gene Polymorphism in Osteoporosis among Guangxi’s Zhuang Chinese
Article Snippet: The gene amplification was carried out in PCR reactions of 20 μL, which included 2 μL of 10×buffer, 2.2 μL of MgCl2 (25 mmol/L), 0.8 μL of dNTPs (10 mmol/L), 1 μL for each upstream and downstream primers (10 μmol/L), 1 μL of template, 0.2 μL of Taq DNA polymerase, and an appropriate amount of double-distilled water to obtain 20 μL volume. .. PCR reaction conditions were as follows: 95 °C for 2 min; 11 cycles of 94 °C for 20 s, 65–0.5 °C/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; 72 °C for 2 min; and 4 °C.PCR products were firstly purified with shrimp alkaline phosphatase (SAP, Promega, Madison, WI, USA) and Exonuclease I (EXO I, Epicentre, Wisconsin, USA) and then were used for the extension reaction with SNaPshot Multiplex kit (Applied Biosystems, Foster, CA, USA). .. The extension product after purification by SAP was sampled on ABI3130xl.

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: Diversity of Pea-Associated F. proliferatum and F. verticillioides Populations Revealed by FUM1 Sequence Analysis and Fumonisin Biosynthesis
Article Snippet: Amplicons were electrophoresed in 1.5% agarose gels (Invitrogen, Carlsbad, CA, USA) with ethidium bromide. .. PCR-amplified DNA fragments were purified for sequence analysis with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following program: 30 min at 37 °C, followed by 15 min at 80 °C. .. Both strands were labeled using the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to Błaszczyk et al. [ ] and the manufacturer’s instructions.

Article Title: Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample
Article Snippet: The PCR primers were designed with the free internet tool Primer3 v.0.4.0 ( http://bioinfo.ut.ee/primer3-0.4.0/ ). .. PCR products were purified by Exo/SAP digestion with Exonuclease I (New England Biolabs, Beverly, MA) and Shrimp Alkaline Phosphatase (Promega, San Diego, USA) and directly sequenced using the ABI PRISM BigDye® Terminator v1.1 Cycle Sequencing Kit and the ABI 3730 sequencing instrument (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. .. All primer sequences and PCR conditions can be obtained on request from the authors.

Article Title: Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species
Article Snippet: Amplicons were electrophoresed in 1.5% agarose gels (Invitrogen, Carlsbad, CA, USA) with ethidium bromide. .. 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.

Article Title: Phylogeography of Eastern Grey Kangaroos, Macropus giganteus, Suggests a Mesic Refugium in Eastern Australia
Article Snippet: PCR reactions were run with the following conditions: initial denaturation at 94°C for 15 minutes, 40 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, followed by a final annealing step at 50°C for 5 minutes and a final extension at 72°C for 10 minutes. .. PCR products were purified by adding 5 U exonuclease I, Escherichia coli (Fermentas, Scoresby) and 0.5 U shrimp alkaline phosphatase (Promega, Hawthorn) and incubating at 37°C for 30 minutes followed by an enzyme inactivation step of 80°C for 15 minutes. .. Following purification, we performed a cycle sequencing reaction by using a 12 μL reaction mixture with 3.0 μL 5x sequencing buffer, 0.5 μL primer, and 1.0 μL BigDye v3.1 (Applied Biosystems, Mulgrave).

Article Title: Functional Variants in NFKBIE and RTKN2 Involved in Activation of the NF-?B Pathway Are Associated with Rheumatoid Arthritis in Japanese
Article Snippet: DNA fragments were amplified with the appropriate primers ( ). .. Purification of PCR products was performed with Exonuclease I (New England Biolabs, Ipswich, MA, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA). .. The amplified DNAs were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), and signals were detected using an ABI 3700 DNA Analyzer (Applied Biosystems).

Article Title: Renal Function and Klotho Gene Polymorphisms among Uygur and Kazak Populations in Xinjiang, China
Article Snippet: Briefly, the SNaPshot reaction was carried out in a 10 μL final volume containing SNaPshot Multiplex Ready Mix (5 μL), primer mix (1 μmol/L), and templates (2 μL). .. The cycling program condition was: denaturation at 96°C for 1 min, followed by 28 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. The extension products were purified by incubation with 1 U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C for 60 min. .. 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 then separated by capillary electrophoresis (ABI PRISM3130 Genetic Analyzer; Applied Biosystems).

Polymerase Chain Reaction:

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: The HSA21 gene EURL/C21ORF91 controls neurogenesis within the cerebral cortex and is implicated in the pathogenesis of Down Syndrome
Article Snippet: Since patients diagnosed with Down Syndrome show clear evidence of abnormal neuronal development, including reduced brain volumes , disorganised cortical layers indicative of aberrant neuronal positioning , and reduced dendritic spine densities on cortical neurons ; we suggest that altered EURL dosage contributes to such neurological deficits in Down Syndrome. .. 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). .. A similar strategy was employed to clone mouse Ccdc85b cDNA from IMAGE:5710206 (Genbank Accession Number BC058411).

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 conditions were; 95°C for 5 min followed by 40 cycles of 95°C for 30 s, an annealing temperature specific to each primer pair (Online Resource 1) for 1 min and 72°C for 1 min. .. 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. .. Sequencing reactions were undertaken by Source Bioscience (Nottingham, UK).

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: 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. .. The probes for LDR are shown in Table .

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: Contribution of DNA repair xeroderma pigmentosum group D genotypes to pancreatic cancer risk in the Chinese Han population
Article Snippet: PCR products were purified using an Exo/SAP protocol (Applied Biosystems, Foster City, CA, USA) according to the manufaccturers instructions. .. The PCR products were labeled by the alkaline phosphatase-labeled streptavidin biotin/exonuclease I (Exo I) method, using 1 U of shrimp alkaline phosphatase (SAP; Promega, Madison, WI, USA) and 1 U of Exo I (New England Biolabs, Beverly, MA, USA). .. The SNaPshot extension reaction system consisted of 5 μL of the SNaPshot multiple reaction kit agents (Applied Biosystems), 2 μL of purified multiple PCR products, and 1 μL of the extension primer mixture, and completing the volume to 10 μL with ultra pure water.

Article Title: Diversity of Pea-Associated F. proliferatum and F. verticillioides Populations Revealed by FUM1 Sequence Analysis and Fumonisin Biosynthesis
Article Snippet: Amplicons were electrophoresed in 1.5% agarose gels (Invitrogen, Carlsbad, CA, USA) with ethidium bromide. .. PCR-amplified DNA fragments were purified for sequence analysis with exonuclease I (Epicentre, Madison, WI, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA) using the following program: 30 min at 37 °C, followed by 15 min at 80 °C. .. Both strands were labeled using the BigDyeTerminator 3.1 kit (Applied Biosystems, Foster City, CA, USA), according to Błaszczyk et al. [ ] and the manufacturer’s instructions.

Article Title: Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample
Article Snippet: The PCR primers were designed with the free internet tool Primer3 v.0.4.0 ( http://bioinfo.ut.ee/primer3-0.4.0/ ). .. PCR products were purified by Exo/SAP digestion with Exonuclease I (New England Biolabs, Beverly, MA) and Shrimp Alkaline Phosphatase (Promega, San Diego, USA) and directly sequenced using the ABI PRISM BigDye® Terminator v1.1 Cycle Sequencing Kit and the ABI 3730 sequencing instrument (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. .. All primer sequences and PCR conditions can be obtained on request from the authors.

Article Title: Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species
Article Snippet: Amplicons were electrophoresed in 1.5% agarose gels (Invitrogen, Carlsbad, CA, USA) with ethidium bromide. .. 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.

Article Title: Phylogeography of Eastern Grey Kangaroos, Macropus giganteus, Suggests a Mesic Refugium in Eastern Australia
Article Snippet: PCR reactions were run with the following conditions: initial denaturation at 94°C for 15 minutes, 40 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, followed by a final annealing step at 50°C for 5 minutes and a final extension at 72°C for 10 minutes. .. PCR products were purified by adding 5 U exonuclease I, Escherichia coli (Fermentas, Scoresby) and 0.5 U shrimp alkaline phosphatase (Promega, Hawthorn) and incubating at 37°C for 30 minutes followed by an enzyme inactivation step of 80°C for 15 minutes. .. Following purification, we performed a cycle sequencing reaction by using a 12 μL reaction mixture with 3.0 μL 5x sequencing buffer, 0.5 μL primer, and 1.0 μL BigDye v3.1 (Applied Biosystems, Mulgrave).

Article Title: Functional Variants in NFKBIE and RTKN2 Involved in Activation of the NF-?B Pathway Are Associated with Rheumatoid Arthritis in Japanese
Article Snippet: DNA fragments were amplified with the appropriate primers ( ). .. Purification of PCR products was performed with Exonuclease I (New England Biolabs, Ipswich, MA, USA) and shrimp alkaline phosphatase (Promega, Madison, WI, USA). .. The amplified DNAs were sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), and signals were detected using an ABI 3700 DNA Analyzer (Applied Biosystems).

Article Title: Renal Function and Klotho Gene Polymorphisms among Uygur and Kazak Populations in Xinjiang, China
Article Snippet: The obtained digestion products were visualized on a 2% agarose gel, and the PCR product was purified by a PCR purification kit (Qiagen, Mildren, Germany). .. The cycling program condition was: denaturation at 96°C for 1 min, followed by 28 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. The extension products were purified by incubation with 1 U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C for 60 min.

Plasmid Preparation:

Article Title: Targeting Mycobacterium tuberculosis Tumor Necrosis Factor Alpha-Downregulating Genes for the Development of Antituberculous Vaccines
Article Snippet: Complementation of the WT gene of interest (driven by the hsp60 promoter) into the attB site of the corresponding M. tuberculosis knockout strain was achieved using the integrating vector pMV361 as previously described ( ). .. 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.

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 diluted ligation product (0.5 μ L) was mixed with 0.5 μ L Liz500 Size Standard and 9 μ L Hi-Di, denatured at 95°C for 5 min, and then loaded in the ABI3730XL sequencer.

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: Contribution of DNA repair xeroderma pigmentosum group D genotypes to pancreatic cancer risk in the Chinese Han population
Article Snippet: PCR primers and extended primers ( ) were designed using the Primer5 software. .. The PCR products were labeled by the alkaline phosphatase-labeled streptavidin biotin/exonuclease I (Exo I) method, using 1 U of shrimp alkaline phosphatase (SAP; Promega, Madison, WI, USA) and 1 U of Exo I (New England Biolabs, Beverly, MA, USA).

Article Title: FGF-2 Gene Polymorphism in Osteoporosis among Guangxi’s Zhuang Chinese
Article Snippet: Primer 5 software was used to design primers, which were synthesized by GENESKY (Shanghai Genesky Biotech Co., Ltd., Shanghai, China). .. PCR reaction conditions were as follows: 95 °C for 2 min; 11 cycles of 94 °C for 20 s, 65–0.5 °C/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; 72 °C for 2 min; and 4 °C.PCR products were firstly purified with shrimp alkaline phosphatase (SAP, Promega, Madison, WI, USA) and Exonuclease I (EXO I, Epicentre, Wisconsin, USA) and then were used for the extension reaction with SNaPshot Multiplex kit (Applied Biosystems, Foster, CA, USA).

Article Title: Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample
Article Snippet: PCR products were purified by Exo/SAP digestion with Exonuclease I (New England Biolabs, Beverly, MA) and Shrimp Alkaline Phosphatase (Promega, San Diego, USA) and directly sequenced using the ABI PRISM BigDye® Terminator v1.1 Cycle Sequencing Kit and the ABI 3730 sequencing instrument (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. .. PCR products were purified by Exo/SAP digestion with Exonuclease I (New England Biolabs, Beverly, MA) and Shrimp Alkaline Phosphatase (Promega, San Diego, USA) and directly sequenced using the ABI PRISM BigDye® Terminator v1.1 Cycle Sequencing Kit and the ABI 3730 sequencing instrument (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions.

Article Title: Renal Function and Klotho Gene Polymorphisms among Uygur and Kazak Populations in Xinjiang, China
Article Snippet: The cycling program condition was: denaturation at 96°C for 1 min, followed by 28 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. The extension products were purified by incubation with 1 U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C for 60 min. .. 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 then separated by capillary electrophoresis (ABI PRISM3130 Genetic Analyzer; Applied Biosystems).

Functional Assay:

Article Title: Contribution of DNA repair xeroderma pigmentosum group D genotypes to pancreatic cancer risk in the Chinese Han population
Article Snippet: XPD codon 751 (rs13181), which is the most studied in the literature, was the functional site that encoded non-synonymous amino acid changes, and was enrolled into this study as well. .. The PCR products were labeled by the alkaline phosphatase-labeled streptavidin biotin/exonuclease I (Exo I) method, using 1 U of shrimp alkaline phosphatase (SAP; Promega, Madison, WI, USA) and 1 U of Exo I (New England Biolabs, Beverly, MA, USA).

Selection:

Article Title: Contribution of DNA repair xeroderma pigmentosum group D genotypes to pancreatic cancer risk in the Chinese Han population
Article Snippet: Paragraph title: SNP selection and genotyping ... The PCR products were labeled by the alkaline phosphatase-labeled streptavidin biotin/exonuclease I (Exo I) method, using 1 U of shrimp alkaline phosphatase (SAP; Promega, Madison, WI, USA) and 1 U of Exo I (New England Biolabs, Beverly, MA, USA).

Article Title: Renal Function and Klotho Gene Polymorphisms among Uygur and Kazak Populations in Xinjiang, China
Article Snippet: Paragraph title: Single-nucleotide polymorphism (SNP) selection and genotyping ... The cycling program condition was: denaturation at 96°C for 1 min, followed by 28 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. The extension products were purified by incubation with 1 U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C for 60 min.

Agarose Gel Electrophoresis:

Article Title: Renal Function and Klotho Gene Polymorphisms among Uygur and Kazak Populations in Xinjiang, China
Article Snippet: The obtained digestion products were visualized on a 2% agarose gel, and the PCR product was purified by a PCR purification kit (Qiagen, Mildren, Germany). .. The cycling program condition was: denaturation at 96°C for 1 min, followed by 28 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. The extension products were purified by incubation with 1 U of shrimp alkaline phosphatase (Promega, Madison, WI) at 37°C for 60 min.

In Vitro:

Article Title: Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA
Article Snippet: The DNA template was digested by DNase I provided with the kit. .. In order to eliminate 5′-triphosphate moieties from in vitro transcribed U1-snRNA that may artificially activate RIG-I , the molecule was treated with shrimp alkaline phosphatase (SAP; Promega, Mannheim, Germany). .. For that purpose, a protocol proven to effectively remove radiolabeled 5′-phosphate ends from in vitro transcribed U1-snRNA by at least 97% was used (data not shown).

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.

Quantitation Assay:

Article Title: Nucleotide Bias Observed with a Short SELEX RNA Aptamer Library
Article Snippet: Paragraph title: High-performance liquid chromatography-based quantitation of NTP concentrations ... The triphosphate groups from the nucleotides (ribonucleotides and deoxynucleotides) were removed using shrimp alkaline phosphatase (Promega, M9910).

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: 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: Since patients diagnosed with Down Syndrome show clear evidence of abnormal neuronal development, including reduced brain volumes , disorganised cortical layers indicative of aberrant neuronal positioning , and reduced dendritic spine densities on cortical neurons ; we suggest that altered EURL dosage contributes to such neurological deficits in Down Syndrome. .. 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). .. A similar strategy was employed to clone mouse Ccdc85b cDNA from IMAGE:5710206 (Genbank Accession Number BC058411).

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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  • 75
    Promega shrimp alkaline phosphatase
    Shrimp Alkaline Phosphatase, supplied by Promega, used in various techniques. Bioz Stars score: 75/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/shrimp alkaline phosphatase/product/Promega
    Average 75 stars, based on 0 article reviews
    Price from $9.99 to $1999.99
    shrimp alkaline phosphatase - by Bioz Stars, 2019-12
    75/100 stars
      Buy from Supplier

    99
    Promega shrimp alkaline phosphatase sap
    EV-A71-derived, double-stranded <t>RNA</t> is a ligand for TLR3 detection. ( A – D ) HEK293 cells were transfected with either a control vector or Flag-tagged TLR3, plus an IFN-β reporter plasmid ( A , C ) or an ISRE reporter plasmid ( B , D ). After 24 h, the transfected cells were either left untreated or infected with EV-A71 (MOI, 0.1) or heat-inactivated EV-A71 (iEV-A71) (MOI, 0.1). After another 40 h, the treated cells were harvested to analyze the IFN-β or ISRE promoter activity ( A , B ). Control RNA (ctrl, 2 µg/mL) and EV-A71-infected RNA (two doses, 1 and 2 µg/mL) harvested from HEK293 cells and EV-A71-infected HEK293 cells, respectively, were used to stimulate the transfected cells by DOTAP lipofection. After another 20 h, the treated cells were harvested to analyze the IFN-β or ISRE promoter activity ( C , D ). ( E ) HEK293 cells were transfected with Flag-tagged TLR3. Control RNA (1 µg/mL) and EV-A71-infected RNA (1µg/mL) were used to stimulate the transfected cells by DOTAP lipofection. After another 20 h, IFN-β in the supernatants was measured using ELISA. ( F ) Like Panel A, HEK293 cells were transfected as indicated. EV-A71-infected RNA left untreated or treated with RNase III or Shrimp Alkanine Phosphatase <t>(SAP)</t> was used to stimulate the transfected cells by DOTAP lipofection. Subsequently, these cells were subjected to luciferase assays. NS: not significant (Student’s t -test). Data are representative of at least two or three independent experiments.
    Shrimp Alkaline Phosphatase Sap, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/shrimp alkaline phosphatase sap/product/Promega
    Average 99 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    shrimp alkaline phosphatase sap - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

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    EV-A71-derived, double-stranded RNA is a ligand for TLR3 detection. ( A – D ) HEK293 cells were transfected with either a control vector or Flag-tagged TLR3, plus an IFN-β reporter plasmid ( A , C ) or an ISRE reporter plasmid ( B , D ). After 24 h, the transfected cells were either left untreated or infected with EV-A71 (MOI, 0.1) or heat-inactivated EV-A71 (iEV-A71) (MOI, 0.1). After another 40 h, the treated cells were harvested to analyze the IFN-β or ISRE promoter activity ( A , B ). Control RNA (ctrl, 2 µg/mL) and EV-A71-infected RNA (two doses, 1 and 2 µg/mL) harvested from HEK293 cells and EV-A71-infected HEK293 cells, respectively, were used to stimulate the transfected cells by DOTAP lipofection. After another 20 h, the treated cells were harvested to analyze the IFN-β or ISRE promoter activity ( C , D ). ( E ) HEK293 cells were transfected with Flag-tagged TLR3. Control RNA (1 µg/mL) and EV-A71-infected RNA (1µg/mL) were used to stimulate the transfected cells by DOTAP lipofection. After another 20 h, IFN-β in the supernatants was measured using ELISA. ( F ) Like Panel A, HEK293 cells were transfected as indicated. EV-A71-infected RNA left untreated or treated with RNase III or Shrimp Alkanine Phosphatase (SAP) was used to stimulate the transfected cells by DOTAP lipofection. Subsequently, these cells were subjected to luciferase assays. NS: not significant (Student’s t -test). Data are representative of at least two or three independent experiments.

    Journal: Viruses

    Article Title: Toll-Like Receptor 3 Is Involved in Detection of Enterovirus A71 Infection and Targeted by Viral 2A Protease

    doi: 10.3390/v10120689

    Figure Lengend Snippet: EV-A71-derived, double-stranded RNA is a ligand for TLR3 detection. ( A – D ) HEK293 cells were transfected with either a control vector or Flag-tagged TLR3, plus an IFN-β reporter plasmid ( A , C ) or an ISRE reporter plasmid ( B , D ). After 24 h, the transfected cells were either left untreated or infected with EV-A71 (MOI, 0.1) or heat-inactivated EV-A71 (iEV-A71) (MOI, 0.1). After another 40 h, the treated cells were harvested to analyze the IFN-β or ISRE promoter activity ( A , B ). Control RNA (ctrl, 2 µg/mL) and EV-A71-infected RNA (two doses, 1 and 2 µg/mL) harvested from HEK293 cells and EV-A71-infected HEK293 cells, respectively, were used to stimulate the transfected cells by DOTAP lipofection. After another 20 h, the treated cells were harvested to analyze the IFN-β or ISRE promoter activity ( C , D ). ( E ) HEK293 cells were transfected with Flag-tagged TLR3. Control RNA (1 µg/mL) and EV-A71-infected RNA (1µg/mL) were used to stimulate the transfected cells by DOTAP lipofection. After another 20 h, IFN-β in the supernatants was measured using ELISA. ( F ) Like Panel A, HEK293 cells were transfected as indicated. EV-A71-infected RNA left untreated or treated with RNase III or Shrimp Alkanine Phosphatase (SAP) was used to stimulate the transfected cells by DOTAP lipofection. Subsequently, these cells were subjected to luciferase assays. NS: not significant (Student’s t -test). Data are representative of at least two or three independent experiments.

    Article Snippet: To digest double-stranded RNA, 4 µg of total RNA was treated with RNaseIII (Epicentre) at the indicated concentration at 37 °C for 30 min. To remove the 5’-triphosphate moiety, 4 µg of total RNA was digested with shrimp alkaline phosphatase (SAP) (Promega) at the indicated concentration at 37 °C for 15 min, and then at 65 °C for 15 min for heat inactivation.

    Techniques: Derivative Assay, Transfection, Plasmid Preparation, Infection, Activity Assay, Enzyme-linked Immunosorbent Assay, Luciferase