pcr based quikchange site directed mutagenesis kit  (Stratagene)

 
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    Structured Review

    Stratagene pcr based quikchange site directed mutagenesis kit
    Pcr Based Quikchange Site Directed Mutagenesis Kit, supplied by Stratagene, used in various techniques. Bioz Stars score: 95/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based quikchange site directed mutagenesis kit/product/Stratagene
    Average 95 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
    pcr based quikchange site directed mutagenesis kit - by Bioz Stars, 2020-02
    95/100 stars

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    Related Articles

    Clone Assay:

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: The COL2A1 sequence spanning –577 to +3,428 bp ( ; ) was cloned into the pGL2-basic (pGL2B) luciferase reporter gene vector (Promega) to generate the pGL2-COL2 construct containing approximately 4 kb of 5′-flanking sequence. .. Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

    Article Title: Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis
    Article Snippet: Paragraph title: Cloning of Golgin-160 ... Mutagenesis of golgin-160 was carried out using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene), and the mutations were confirmed by dideoxy sequencing.

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: The plasmid pBP1 was digested with Nhe I and Xho I and the excised gene was cloned into the similarly restricted expression vector pET-21a such that the recombinant protein contained an N-terminal His6 tag. .. The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.

    Article Title: Mutational Analysis Reveals Distinct Features of the Nox4-p22phox Complex * Complex * S⃞
    Article Snippet: Amino acid exchange mutants W6/9F, W6/9A, W6/9R, W6/9D, H72H, R90Q, P156Q, and Y121H and various lysine exchange mutants in regions aa 32-35 and aa 98-102 were generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. .. Mouse Nox4 was a gift of Dr. B. Banfi; mouse p22phox was cloned from mouse macrophages.

    Article Title: Nuclear Factor Erythroid 2-like 2 Regulates Expression of Inositol 1,4,5-trisphosphate Receptor, Type 3 and Calcium Signaling in Cholangiocytes
    Article Snippet: The 2120 bp DNA fragment containing the human ITPR3 proximal promoter region was cloned into a pGL4.17 luciferase vector (Promega, Madison, WI). .. The MARE reporter construct was mutated using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene; Cedar creek, TX).

    Amplification:

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: The resulting DNA fragment was cloned into pGEM T-easy, generating pBP1, and sequenced to ensure that no mutations had occurred during amplification. .. The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.

    Article Title: Nuclear Factor Erythroid 2-like 2 Regulates Expression of Inositol 1,4,5-trisphosphate Receptor, Type 3 and Calcium Signaling in Cholangiocytes
    Article Snippet: A 2.1 kb DNA fragment containing the human ITPR3 proximal promoter region was amplified by PCR using a BAC Clone CH230-6B20, accession no. obtained from Childrens Hospital of Oakland Research Institute (BacPac Resources, Oakland, CA) as a template. .. The MARE reporter construct was mutated using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene; Cedar creek, TX).

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: The amplified products were digested with EcoRI and HindIII (New England BioLabs, Ipswich, MA) and inserted into the EcoRI and HindIII restriction sites of a FLAG-tagged expression vector, pCMV-Tag2B (Agilent Technologies, Santa Clara, CA). .. Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.

    Construct:

    Article Title: Ring Finger Protein 149 Is an E3 Ubiquitin Ligase Active on Wild-type v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) *
    Article Snippet: .. The mutant constructs BRAFV600E and RNF149H289A , in which Val-600 was replaced with glutamine and His-289 was replaced with alanine, respectively, were created using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene). .. Lipofectamine 2000 (Invitrogen) was used for transient transfections.

    Article Title: Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7
    Article Snippet: Both amino- and carboxyl-terminal FLAG-epitope-tagged 16E7 constructs were generated by PCR. .. The 16E7 K60,97R mutant was generated using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.).

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: .. Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). .. Luciferase reporter plasmids were prepared for transfection using the EndoFree plasmid maxi kit (Qiagen).

    Article Title: Nuclear Factor Erythroid 2-like 2 Regulates Expression of Inositol 1,4,5-trisphosphate Receptor, Type 3 and Calcium Signaling in Cholangiocytes
    Article Snippet: .. The MARE reporter construct was mutated using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene; Cedar creek, TX). ..

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: .. Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. .. DNA was amplified by PCR with complimentary primer pairs and wild type expression plasmid pCMV-LANA as the template.

    Luciferase:

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: Paragraph title: Luciferase reporter constructs and expression plasmids ... Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

    Article Title: Nuclear Factor Erythroid 2-like 2 Regulates Expression of Inositol 1,4,5-trisphosphate Receptor, Type 3 and Calcium Signaling in Cholangiocytes
    Article Snippet: The 2120 bp DNA fragment containing the human ITPR3 proximal promoter region was cloned into a pGL4.17 luciferase vector (Promega, Madison, WI). .. The MARE reporter construct was mutated using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene; Cedar creek, TX).

    Activity Assay:

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: Mutation of the translation start site ATG to CTG in the pGL2-COL2 construct did not increase the activity indicating that splicing occurred correctly and did not interfere with translation of the luciferase reporter. .. Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

    Expressing:

    Article Title: Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7
    Article Snippet: The 16E7-FLAG PCR product was subcloned into the Hin dIII and Not I sites of the pcDNA3 expression plasmid (Invitrogen). .. The 16E7 K60,97R mutant was generated using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.).

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: Paragraph title: Luciferase reporter constructs and expression plasmids ... Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: Paragraph title: 2.2. Protein expression and purification ... The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.

    Article Title: Mutational Analysis Reveals Distinct Features of the Nox4-p22phox Complex * Complex * S⃞
    Article Snippet: Plasmids and Transfections —Human Nox4 expression plasmid pcDNA3.0 Nox4, p22phox WT, and p22phox truncation and deletion mutants N5, N11, I-6, I-26, C130, C131, C141, and C171 in pcDNA3.1+ (Zeo) were described earlier ( ). .. Amino acid exchange mutants W6/9F, W6/9A, W6/9R, W6/9D, H72H, R90Q, P156Q, and Y121H and various lysine exchange mutants in regions aa 32-35 and aa 98-102 were generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol.

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: .. Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. .. DNA was amplified by PCR with complimentary primer pairs and wild type expression plasmid pCMV-LANA as the template.

    Modification:

    Article Title: Ring Finger Protein 149 Is an E3 Ubiquitin Ligase Active on Wild-type v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) *
    Article Snippet: The human colon cancer cell lines, HCT-116 and RKO, and the human embryonic kidney line, 293T, were cultured in 5% CO2 at 37 °C in high-glucose Dulbecco's modified Eagle's medium (DMEM; GIBCO). .. The mutant constructs BRAFV600E and RNF149H289A , in which Val-600 was replaced with glutamine and His-289 was replaced with alanine, respectively, were created using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene).

    Article Title: Mutational Analysis Reveals Distinct Features of the Nox4-p22phox Complex * Complex * S⃞
    Article Snippet: Human lung carcinoma cells H661 (ATCC® number HTB-183™) were cultivated in RPMI 1640, and HEK-EV and HEK-Nox4-11 ( ) were grown in Dulbecco's modified Eagle's medium. .. Amino acid exchange mutants W6/9F, W6/9A, W6/9R, W6/9D, H72H, R90Q, P156Q, and Y121H and various lysine exchange mutants in regions aa 32-35 and aa 98-102 were generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol.

    Countercurrent Chromatography:

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: Full-length ORF73 (LANA) gene was amplified by PCR with specific primers: ORF73F- CAT GAA TTC ATG GCG CCC CCG GGA ATG CGC, and ORF73R- CAT AAG CTT TGT CAT TTC CTG TGG AGA GTC containing EcoRI and HindIII restriction sites at the 5' and 3' ends, respectively. .. Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.

    Transfection:

    Article Title: Ring Finger Protein 149 Is an E3 Ubiquitin Ligase Active on Wild-type v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) *
    Article Snippet: Paragraph title: Cell Culture, Plasmids, and Transfection ... The mutant constructs BRAFV600E and RNF149H289A , in which Val-600 was replaced with glutamine and His-289 was replaced with alanine, respectively, were created using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene).

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). .. Luciferase reporter plasmids were prepared for transfection using the EndoFree plasmid maxi kit (Qiagen).

    Article Title: Mutational Analysis Reveals Distinct Features of the Nox4-p22phox Complex * Complex * S⃞
    Article Snippet: Plasmids and Transfections —Human Nox4 expression plasmid pcDNA3.0 Nox4, p22phox WT, and p22phox truncation and deletion mutants N5, N11, I-6, I-26, C130, C131, C141, and C171 in pcDNA3.1+ (Zeo) were described earlier ( ). .. Amino acid exchange mutants W6/9F, W6/9A, W6/9R, W6/9D, H72H, R90Q, P156Q, and Y121H and various lysine exchange mutants in regions aa 32-35 and aa 98-102 were generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol.

    Ligation:

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: Deletion constructs containing –577/+125 bp, –403/+125 bp, –131/+125 bp, –90/+125 bp were prepared by restriction enzyme digestion of pGL2-COL2 at PstI, NheI, NcoI, ApaI, and PvuII sites, respectively, followed by ligation. .. Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

    Cell Culture:

    Article Title: Ring Finger Protein 149 Is an E3 Ubiquitin Ligase Active on Wild-type v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) *
    Article Snippet: Paragraph title: Cell Culture, Plasmids, and Transfection ... The mutant constructs BRAFV600E and RNF149H289A , in which Val-600 was replaced with glutamine and His-289 was replaced with alanine, respectively, were created using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene).

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. .. To express the recombinant CBM22-1–GH10 E337A protein, E. coli strain BL21 (DE3) harbouring the pET-21a plasmid was cultured in Luria–Bertani broth at 310 K to mid-exponential phase ( A 550 = 0.6).

    Article Title: Mutational Analysis Reveals Distinct Features of the Nox4-p22phox Complex * Complex * S⃞
    Article Snippet: Cell Lines and Cell Culture —CHO-K1 cells were originally obtained from the American Type Culture Collection (Manassas, VA) (CRL-1651). .. Amino acid exchange mutants W6/9F, W6/9A, W6/9R, W6/9D, H72H, R90Q, P156Q, and Y121H and various lysine exchange mutants in regions aa 32-35 and aa 98-102 were generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol.

    Generated:

    Article Title: Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7
    Article Snippet: .. The 16E7 K60,97R mutant was generated using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). .. All primer sequences used in subcloning and site-directed mutagenesis are available upon request.

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: .. Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). .. Luciferase reporter plasmids were prepared for transfection using the EndoFree plasmid maxi kit (Qiagen).

    Article Title: Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis
    Article Snippet: Cloning of Golgin-160 A cDNA clone corresponding to the open reading frame of golgin-160 and flanked by XbaI sites was generated by reverse transcriptase–PCR from human liver and subcloned into the XbaI site of pBlueScript SK+. .. Mutagenesis of golgin-160 was carried out using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene), and the mutations were confirmed by dideoxy sequencing.

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: .. The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. .. The primers used to generate the nucleic acid mutant were GACGTTGTAAAT GCG GCAGTAAGTGATGATGC and GCATCATCACTTACTGC CGC ATTTACAACGTC (nucleotide substitutions in bold).

    Article Title: Mutational Analysis Reveals Distinct Features of the Nox4-p22phox Complex * Complex * S⃞
    Article Snippet: .. Amino acid exchange mutants W6/9F, W6/9A, W6/9R, W6/9D, H72H, R90Q, P156Q, and Y121H and various lysine exchange mutants in regions aa 32-35 and aa 98-102 were generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. .. Mouse Nox4 was a gift of Dr. B. Banfi; mouse p22phox was cloned from mouse macrophages.

    DNA Sequencing:

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). .. The sequences of all constructs were confirmed by DNA sequencing performed at the Beth Israel Deaconess Medical Center DNA sequencing facility using the ABI Prism BigDye™Primer Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and the Automatic DNA Sequencer Model 373A (Applied Biosystems).

    Polymerase Chain Reaction:

    Article Title: Ring Finger Protein 149 Is an E3 Ubiquitin Ligase Active on Wild-type v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) *
    Article Snippet: .. The mutant constructs BRAFV600E and RNF149H289A , in which Val-600 was replaced with glutamine and His-289 was replaced with alanine, respectively, were created using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene). .. Lipofectamine 2000 (Invitrogen) was used for transient transfections.

    Article Title: The Active Site of a Carbohydrate Esterase Displays Divergent Catalytic and Noncatalytic Binding FunctionsGene Sharing Yields an Enzyme with Two Binding Sites in One Subunit
    Article Snippet: .. Site-directed mutagenesis was carried out using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, using pCtCE2 or pCjCE2A as the template and primers pairs that are listed in . ..

    Article Title: Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7
    Article Snippet: .. The 16E7 K60,97R mutant was generated using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). .. All primer sequences used in subcloning and site-directed mutagenesis are available upon request.

    Article Title: An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins
    Article Snippet: .. Site-directed mutagenesis was conducted using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, using the plasmid encoding BACCELL_00875 and BT3687 as the template and appropriate primer pairs. ..

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: .. Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). .. Luciferase reporter plasmids were prepared for transfection using the EndoFree plasmid maxi kit (Qiagen).

    Article Title: Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis
    Article Snippet: .. Mutagenesis of golgin-160 was carried out using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene), and the mutations were confirmed by dideoxy sequencing. .. In Vitro Cleavage of [35 S]Methionine-labeled Substrates Generated by In Vitro Transcription/Translation [35 S]methionine-labeled golgin-160 and golgin-160 mutants were generated by coupled in vitro transcription/translation ( ).

    Article Title: The Structure and Function of an Arabinan-specific ?-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases *
    Article Snippet: .. Site-directed mutagenesis was conducted using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, using pAC12 as the template and the primer pairs listed in . .. The substrates 4-nitrophenyl-α- l -arabinofuranoside (4NPA) and sugar beet arabinan were purchased from Sigma and Megazyme, respectively.

    Article Title: Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a carbohydrate-binding module
    Article Snippet: .. The mutant protein, which was designated Ct CBM3acipA-M5 , was created with a PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene) as described previously ( ). .. Circular Dichroism (CD) spectroscopy CD spectra were recorded with a JASCO 810 spectropolarimeter.

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: .. The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. .. The primers used to generate the nucleic acid mutant were GACGTTGTAAAT GCG GCAGTAAGTGATGATGC and GCATCATCACTTACTGC CGC ATTTACAACGTC (nucleotide substitutions in bold).

    Article Title: Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast
    Article Snippet: .. Mutations in EFT2 were created utilizing the PCR based QuikChange Site-Directed Mutagenesis Kit (Stratagene). pTKB501 (eEF2HA , provided by Dr M. Justice, Merck Research Laboratories) and pTKB612 [eEF2HIS , ( )] were utilized as the templates for PCR. pTKB658 ( eEF 2 V 488 A HIS ) and pTKB696 ( eEF 2 V 488 A HA ) were created using primers 5′V488A (5′-CTCTGTCTCTCCAGCTGTGCAAGTCGCTGTCG-3′) and 3′V488A (5′- CGACACCGACTTGCACAGCAGGAGAGACAGAG-3′). pTKB701 ( eEF 2 H 699 K HA ) and pTKB704 ( eEF 2 H 699 K HIS ) were created using primers 5′H699K (5′-CATGCCGATGCTATCAAGAGAGGTGGTGGTCAAATC-3′) and 3′H699K (5′-GATTTGACCACCACCTCCCTTGATAGCATCGGCATG-3′). .. All mutations were confirmed by restriction digestion and DNA sequence analysis.

    Article Title: Modulation of Endogenous Indole-3-Acetic Acid Biosynthesis in Bacteroids within Medicago sativa Nodules
    Article Snippet: .. Site-directed mutagenesis of the promintron consensus sequences was performed in two steps by using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene). ..

    Article Title: Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function
    Article Snippet: .. Site-directed mutagenesis was carried out by using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) by using appropriate plasmids as templates and primers pairs that are listed in . ..

    Article Title: Nuclear Factor Erythroid 2-like 2 Regulates Expression of Inositol 1,4,5-trisphosphate Receptor, Type 3 and Calcium Signaling in Cholangiocytes
    Article Snippet: .. The MARE reporter construct was mutated using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene; Cedar creek, TX). ..

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: .. Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. .. DNA was amplified by PCR with complimentary primer pairs and wild type expression plasmid pCMV-LANA as the template.

    Binding Assay:

    Article Title: Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a carbohydrate-binding module
    Article Snippet: Five residues of the crystalline cellulose binding site of Ct CBM3acipA were replaced with alanine (D56A, H57A, Y67A, R112A, W118A) in order to block recognition of crystalline cellulose. .. The mutant protein, which was designated Ct CBM3acipA-M5 , was created with a PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene) as described previously ( ).

    Subcloning:

    Article Title: Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7
    Article Snippet: The 16E7 K60,97R mutant was generated using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). .. All primer sequences used in subcloning and site-directed mutagenesis are available upon request.

    Mutagenesis:

    Article Title: Ring Finger Protein 149 Is an E3 Ubiquitin Ligase Active on Wild-type v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) *
    Article Snippet: .. The mutant constructs BRAFV600E and RNF149H289A , in which Val-600 was replaced with glutamine and His-289 was replaced with alanine, respectively, were created using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene). .. Lipofectamine 2000 (Invitrogen) was used for transient transfections.

    Article Title: The Active Site of a Carbohydrate Esterase Displays Divergent Catalytic and Noncatalytic Binding FunctionsGene Sharing Yields an Enzyme with Two Binding Sites in One Subunit
    Article Snippet: .. Site-directed mutagenesis was carried out using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, using pCtCE2 or pCjCE2A as the template and primers pairs that are listed in . ..

    Article Title: Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7
    Article Snippet: .. The 16E7 K60,97R mutant was generated using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.). .. All primer sequences used in subcloning and site-directed mutagenesis are available upon request.

    Article Title: An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins
    Article Snippet: .. Site-directed mutagenesis was conducted using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, using the plasmid encoding BACCELL_00875 and BT3687 as the template and appropriate primer pairs. ..

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: .. Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). .. Luciferase reporter plasmids were prepared for transfection using the EndoFree plasmid maxi kit (Qiagen).

    Article Title: Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis
    Article Snippet: .. Mutagenesis of golgin-160 was carried out using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene), and the mutations were confirmed by dideoxy sequencing. .. In Vitro Cleavage of [35 S]Methionine-labeled Substrates Generated by In Vitro Transcription/Translation [35 S]methionine-labeled golgin-160 and golgin-160 mutants were generated by coupled in vitro transcription/translation ( ).

    Article Title: The Structure and Function of an Arabinan-specific ?-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases *
    Article Snippet: .. Site-directed mutagenesis was conducted using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, using pAC12 as the template and the primer pairs listed in . .. The substrates 4-nitrophenyl-α- l -arabinofuranoside (4NPA) and sugar beet arabinan were purchased from Sigma and Megazyme, respectively.

    Article Title: Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a carbohydrate-binding module
    Article Snippet: .. The mutant protein, which was designated Ct CBM3acipA-M5 , was created with a PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene) as described previously ( ). .. Circular Dichroism (CD) spectroscopy CD spectra were recorded with a JASCO 810 spectropolarimeter.

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: .. The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. .. The primers used to generate the nucleic acid mutant were GACGTTGTAAAT GCG GCAGTAAGTGATGATGC and GCATCATCACTTACTGC CGC ATTTACAACGTC (nucleotide substitutions in bold).

    Article Title: Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast
    Article Snippet: .. Mutations in EFT2 were created utilizing the PCR based QuikChange Site-Directed Mutagenesis Kit (Stratagene). pTKB501 (eEF2HA , provided by Dr M. Justice, Merck Research Laboratories) and pTKB612 [eEF2HIS , ( )] were utilized as the templates for PCR. pTKB658 ( eEF 2 V 488 A HIS ) and pTKB696 ( eEF 2 V 488 A HA ) were created using primers 5′V488A (5′-CTCTGTCTCTCCAGCTGTGCAAGTCGCTGTCG-3′) and 3′V488A (5′- CGACACCGACTTGCACAGCAGGAGAGACAGAG-3′). pTKB701 ( eEF 2 H 699 K HA ) and pTKB704 ( eEF 2 H 699 K HIS ) were created using primers 5′H699K (5′-CATGCCGATGCTATCAAGAGAGGTGGTGGTCAAATC-3′) and 3′H699K (5′-GATTTGACCACCACCTCCCTTGATAGCATCGGCATG-3′). .. All mutations were confirmed by restriction digestion and DNA sequence analysis.

    Article Title: Modulation of Endogenous Indole-3-Acetic Acid Biosynthesis in Bacteroids within Medicago sativa Nodules
    Article Snippet: .. Site-directed mutagenesis of the promintron consensus sequences was performed in two steps by using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene). ..

    Article Title: Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function
    Article Snippet: .. Site-directed mutagenesis was carried out by using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) by using appropriate plasmids as templates and primers pairs that are listed in . ..

    Article Title: Nuclear Factor Erythroid 2-like 2 Regulates Expression of Inositol 1,4,5-trisphosphate Receptor, Type 3 and Calcium Signaling in Cholangiocytes
    Article Snippet: .. The MARE reporter construct was mutated using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene; Cedar creek, TX). ..

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: .. Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol. .. DNA was amplified by PCR with complimentary primer pairs and wild type expression plasmid pCMV-LANA as the template.

    Microelectrode Array:

    Article Title: Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis
    Article Snippet: We do not believe that the 5-bp insertion was introduced while amplifying the cDNA clone by PCR, as the identical 5-bp insertion is present in a human EST sequence (AL044812) and in the mouse Mea-2 protein ( ). .. Mutagenesis of golgin-160 was carried out using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene), and the mutations were confirmed by dideoxy sequencing.

    Purification:

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: Paragraph title: 2.2. Protein expression and purification ... The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.

    Sequencing:

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: The COL2A1 sequence spanning –577 to +3,428 bp ( ; ) was cloned into the pGL2-basic (pGL2B) luciferase reporter gene vector (Promega) to generate the pGL2-COL2 construct containing approximately 4 kb of 5′-flanking sequence. .. Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

    Article Title: Caspase-2 Is Localized at the Golgi Complex and Cleaves Golgin-160 during Apoptosis
    Article Snippet: .. Mutagenesis of golgin-160 was carried out using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene), and the mutations were confirmed by dideoxy sequencing. .. In Vitro Cleavage of [35 S]Methionine-labeled Substrates Generated by In Vitro Transcription/Translation [35 S]methionine-labeled golgin-160 and golgin-160 mutants were generated by coupled in vitro transcription/translation ( ).

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. .. The mutated DNA sequence was sequenced by MWG (Germany) to ensure that only the appropriate mutation had been incorporated into the nucleic acid.

    Article Title: Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast
    Article Snippet: Mutations in EFT2 were created utilizing the PCR based QuikChange Site-Directed Mutagenesis Kit (Stratagene). pTKB501 (eEF2HA , provided by Dr M. Justice, Merck Research Laboratories) and pTKB612 [eEF2HIS , ( )] were utilized as the templates for PCR. pTKB658 ( eEF 2 V 488 A HIS ) and pTKB696 ( eEF 2 V 488 A HA ) were created using primers 5′V488A (5′-CTCTGTCTCTCCAGCTGTGCAAGTCGCTGTCG-3′) and 3′V488A (5′- CGACACCGACTTGCACAGCAGGAGAGACAGAG-3′). pTKB701 ( eEF 2 H 699 K HA ) and pTKB704 ( eEF 2 H 699 K HIS ) were created using primers 5′H699K (5′-CATGCCGATGCTATCAAGAGAGGTGGTGGTCAAATC-3′) and 3′H699K (5′-GATTTGACCACCACCTCCCTTGATAGCATCGGCATG-3′). .. All mutations were confirmed by restriction digestion and DNA sequence analysis.

    Article Title: Modulation of Endogenous Indole-3-Acetic Acid Biosynthesis in Bacteroids within Medicago sativa Nodules
    Article Snippet: Paragraph title: Site-directed mutagenesis of the promintron sequence. ... Site-directed mutagenesis of the promintron consensus sequences was performed in two steps by using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene).

    Article Title: Nuclear Factor Erythroid 2-like 2 Regulates Expression of Inositol 1,4,5-trisphosphate Receptor, Type 3 and Calcium Signaling in Cholangiocytes
    Article Snippet: MatInspector ( ) was used to analyze the promoter sequence, and a putative MARE was identified. .. The MARE reporter construct was mutated using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene; Cedar creek, TX).

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: Aspartate-to-alanine mutations were made at amino acid position 53 (GAC- > GCC) and 878 (GAT- > GCT) of the BCBL-1 LANA amino acid sequence (GENBANK accession number U93872). .. Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.

    Blocking Assay:

    Article Title: Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a carbohydrate-binding module
    Article Snippet: Five residues of the crystalline cellulose binding site of Ct CBM3acipA were replaced with alanine (D56A, H57A, Y67A, R112A, W118A) in order to block recognition of crystalline cellulose. .. The mutant protein, which was designated Ct CBM3acipA-M5 , was created with a PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene) as described previously ( ).

    Spectroscopy:

    Article Title: Recognition of xyloglucan by the crystalline cellulose-binding site of a family 3a carbohydrate-binding module
    Article Snippet: The mutant protein, which was designated Ct CBM3acipA-M5 , was created with a PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene) as described previously ( ). .. Circular Dichroism (CD) spectroscopy CD spectra were recorded with a JASCO 810 spectropolarimeter.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: Full-length ORF73 (LANA) gene was amplified by PCR with specific primers: ORF73F- CAT GAA TTC ATG GCG CCC CCG GGA ATG CGC, and ORF73R- CAT AAG CTT TGT CAT TTC CTG TGG AGA GTC containing EcoRI and HindIII restriction sites at the 5' and 3' ends, respectively. .. Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.

    Plasmid Preparation:

    Article Title: Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7
    Article Snippet: The 16E7-FLAG PCR product was subcloned into the Hin dIII and Not I sites of the pcDNA3 expression plasmid (Invitrogen). .. The 16E7 K60,97R mutant was generated using a PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, Calif.).

    Article Title: An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins
    Article Snippet: .. Site-directed mutagenesis was conducted using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, using the plasmid encoding BACCELL_00875 and BT3687 as the template and appropriate primer pairs. ..

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: The COL2A1 sequence spanning –577 to +3,428 bp ( ; ) was cloned into the pGL2-basic (pGL2B) luciferase reporter gene vector (Promega) to generate the pGL2-COL2 construct containing approximately 4 kb of 5′-flanking sequence. .. Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: The plasmid pBP1 was digested with Nhe I and Xho I and the excised gene was cloned into the similarly restricted expression vector pET-21a such that the recombinant protein contained an N-terminal His6 tag. .. The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.

    Article Title: Mutational Analysis Reveals Distinct Features of the Nox4-p22phox Complex * Complex * S⃞
    Article Snippet: Plasmids and Transfections —Human Nox4 expression plasmid pcDNA3.0 Nox4, p22phox WT, and p22phox truncation and deletion mutants N5, N11, I-6, I-26, C130, C131, C141, and C171 in pcDNA3.1+ (Zeo) were described earlier ( ). .. Amino acid exchange mutants W6/9F, W6/9A, W6/9R, W6/9D, H72H, R90Q, P156Q, and Y121H and various lysine exchange mutants in regions aa 32-35 and aa 98-102 were generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol.

    Article Title: Nuclear Factor Erythroid 2-like 2 Regulates Expression of Inositol 1,4,5-trisphosphate Receptor, Type 3 and Calcium Signaling in Cholangiocytes
    Article Snippet: The 2120 bp DNA fragment containing the human ITPR3 proximal promoter region was cloned into a pGL4.17 luciferase vector (Promega, Madison, WI). .. The MARE reporter construct was mutated using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene; Cedar creek, TX).

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: The amplified products were digested with EcoRI and HindIII (New England BioLabs, Ipswich, MA) and inserted into the EcoRI and HindIII restriction sites of a FLAG-tagged expression vector, pCMV-Tag2B (Agilent Technologies, Santa Clara, CA). .. Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.

    Positron Emission Tomography:

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: The plasmid pBP1 was digested with Nhe I and Xho I and the excised gene was cloned into the similarly restricted expression vector pET-21a such that the recombinant protein contained an N-terminal His6 tag. .. The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.

    In Vitro:

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: Paragraph title: Production of FLAG-tagged LANA and in vitro caspase cleavage of FLAG-LANA ... Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.

    BAC Assay:

    Article Title: Nuclear Factor Erythroid 2-like 2 Regulates Expression of Inositol 1,4,5-trisphosphate Receptor, Type 3 and Calcium Signaling in Cholangiocytes
    Article Snippet: A 2.1 kb DNA fragment containing the human ITPR3 proximal promoter region was amplified by PCR using a BAC Clone CH230-6B20, accession no. obtained from Childrens Hospital of Oakland Research Institute (BacPac Resources, Oakland, CA) as a template. .. The MARE reporter construct was mutated using the PCR-based QuikChange™ site-directed mutagenesis kit (Stratagene; Cedar creek, TX).

    CTG Assay:

    Article Title: ESE-1 Is a Potent Repressor of Type II Collagen Gene (COL2A1) Transcription In Human Chondrocytes
    Article Snippet: Mutation of the translation start site ATG to CTG in the pGL2-COL2 construct did not increase the activity indicating that splicing occurred correctly and did not interfere with translation of the luciferase reporter. .. Mutations in ETS sites in the pGL2-COL2 construct were generated using the PCR-based QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

    Article Title: Identification of Caspase Cleavage Sites in KSHV Latency-Associated Nuclear Antigen and Their Effects on Caspase-Related Host Defense Responses
    Article Snippet: Full-length ORF73 (LANA) gene was amplified by PCR with specific primers: ORF73F- CAT GAA TTC ATG GCG CCC CCG GGA ATG CGC, and ORF73R- CAT AAG CTT TGT CAT TTC CTG TGG AGA GTC containing EcoRI and HindIII restriction sites at the 5' and 3' ends, respectively. .. Plasmids expressing mutated caspase cleavage sites of LANA were constructed using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.

    Recombinant:

    Article Title: Purification, crystallization and crystallographic analysis of Clostridium thermocellum endo-1,4-?-d-xylanase 10B in complex with xylohexaose
    Article Snippet: The plasmid pBP1 was digested with Nhe I and Xho I and the excised gene was cloned into the similarly restricted expression vector pET-21a such that the recombinant protein contained an N-terminal His6 tag. .. The CBM22-1–GH10 nucleophile mutant (Glu→Ala), termed CBM22-1–GH10 E337A, was generated using the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions.

    Article Title: Dominant-negative mutant phenotypes and the regulation of translation elongation factor 2 levels in yeast
    Article Snippet: DNA manipulations and mutagenesis Recombinant DNA techniques were performed as described ( ). .. Mutations in EFT2 were created utilizing the PCR based QuikChange Site-Directed Mutagenesis Kit (Stratagene). pTKB501 (eEF2HA , provided by Dr M. Justice, Merck Research Laboratories) and pTKB612 [eEF2HIS , ( )] were utilized as the templates for PCR. pTKB658 ( eEF 2 V 488 A HIS ) and pTKB696 ( eEF 2 V 488 A HA ) were created using primers 5′V488A (5′-CTCTGTCTCTCCAGCTGTGCAAGTCGCTGTCG-3′) and 3′V488A (5′- CGACACCGACTTGCACAGCAGGAGAGACAGAG-3′). pTKB701 ( eEF 2 H 699 K HA ) and pTKB704 ( eEF 2 H 699 K HIS ) were created using primers 5′H699K (5′-CATGCCGATGCTATCAAGAGAGGTGGTGGTCAAATC-3′) and 3′H699K (5′-GATTTGACCACCACCTCCCTTGATAGCATCGGCATG-3′).

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    Stratagene pcr based site directed mutagenesis the quikchange site directed mutagenesis kit stratagene la jolla ca
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