Log In Sign up
The largest database of trusted experimental protocols

Geneart site directed mutagenesis system

Manufactured by Thermo Fisher Scientific
Visit Supplier
Sourced in United States

The GeneArt™ Site-Directed Mutagenesis System is a tool used for introducing specific modifications or changes into DNA sequences. It provides a simple and efficient way to generate desired mutations within a target gene or plasmid.

Automatically generated - may contain errors

Lab products found in correlation

Dual luciferase reporter assay system, by Promega (15 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (11 mentions) Sequencher 4, by Gene Codes (8 mentions) Pmirglo luciferase vector, by Promega (7 mentions) Dual luciferase assay system, by Promega (6 mentions) Pmirglo dual luciferase mirna target expression vector, by Promega (6 mentions) Ptarget vector, by Promega (5 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions) Sequencer 4.8 program, by Gene Codes (4 mentions) Onetaq 2 master mix with standard buffer, by New England Biolabs (4 mentions) Full length human s6k1 cdna in pcmv6 entry vector, by OriGene (3 mentions) Pcmv6 entry vector, by OriGene (3 mentions) Pdonr207, by Thermo Fisher Scientific (3 mentions) Psicheck 2 vector, by Promega (3 mentions) E coli top10 cells, by Thermo Fisher Scientific (3 mentions) Pcdna3.1d v5 his topo vector, by Thermo Fisher Scientific (3 mentions) Rneasy mini kit, by Qiagen (3 mentions) Pmirglo vector, by Promega (3 mentions) Luciferase reporter assay system, by Promega (3 mentions) Recombinant active s6k132, by Cell Signaling Technology (2 mentions)

Close spelling variants of this product

GeneArt (908 citations) GeneArt™ Site-Directed Mutagenesis PLUS System (27 citations) Site-directed mutagenesis (14 citations) GeneArt Site-Directed Mutagenesis System kit (10 citations) Site-Directed Mutagenesis System (9 citations) GeneArt Site-Directed Mutagenesis (2 citations)

201 protocols using geneart site directed mutagenesis system

1

Cloning and Mutagenesis of Sar1a and Sec12

Check if the same lab product or an alternative is used in the 5 most similar protocols
The Sar1a and Sec12 cDNA clones from swine and cattle were purchased from Origene (sSar1: NM_001031786; cSar1: XM_005226384; sSec12: XM_003125297; cSec12: NM_001076875). They were inserted into pcDNA-3.1(+) with a FLAG tag at the N-terminus. The sSar1 mutant constructs, including H79G, T39N, and QTTG (156-QTTG-159 to AAAA), were generated using the GENEART site-directed mutagenesis system (Invitrogen) with the indicated primers, while D198 was replaced with alanine using standard PCR with the primers BamHI-sSar1-F and XhoI-msSar1-D198A-R. The mutation of I41A for sSec12 was also performed with a GENEART site-directed mutagenesis system (Invitrogen).
Lee H.W., Jiang Y.F., Chang H.W, & Cheng I.C. (2022). Foot-and-Mouth Disease Virus 3A Hijacks Sar1 and Sec12 for ER Remodeling in a COPII-Independent Manner. Viruses, 14(4), 839.
+ Open protocol
+ Expand
2

Purification and Characterization of S6K1 Variants

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Recombinant, His-tagged, wild-type and Ser886-to-Ala (S886A) mutant linker proteins spanning Pro683 to Asn1023 of human EPRS were expressed and purified as described (Arif et al., 2011 (link); Arif et al., 2009 (link)). Recombinant, active, full-length S6K1 (rS6K1FL, 1502 aa plus NLS) was purchased from R&D Systems and Cell Signaling (Kozma et al., 1993 (link)). Full-length human S6K1 cDNA in pCMV6-Entry vector was purchased from Origene and recloned, deleting the 23-aa N-terminus nuclear localization sequence (NLS) and adding an in-frame upstream 6-His tag and downstream C-terminus Myc-tag in pcDNA3. Specific mutations were introduced using appropriate mutation-bearing primers and GENEART Site-Directed Mutagenesis System (Invitrogen). Recombinant, wild-type and mutant NLS-deleted S6K1 with N-terminus His- and C-terminus Myc-tag were generated by in vitro translation using wheat germ extract system (Promega), and purified by Ni-affinity chromatography (Thermo Scientific Pierce).
Arif A., Jia J., Willard B., Li X, & Fox P.L. (2019). Multisite phosphorylation of S6K1 directs a kinase phospho-code that determines substrate selection. Molecular cell, 73(3), 446-457.e6.
+ Open protocol
+ Expand
3

Cloning and Mutagenesis of AYVV Coat Protein

Check if the same lab product or an alternative is used in the 5 most similar protocols
Oligonucleotides KS37 and KS38 (Supplementary Table 2) were used with pHNBin419 DNA as template and Phusion polymerase (New England Biolabs) for PCR to obtain DNA encoding the AYVV CP that was subsequently cloned via the BP reaction into pDONR207 (Invitrogen). A sequenced-verified clone was transferred via the Gateway LR reaction into pEAQ-HT-DEST131 (link) to yield pEAQ-HT-D1-AYVVCP. The Agilent “QuikChange Primer Design” website, http://www.genomics.agilent.com/primerDesignProgram.jsp was used to design primer pairs to introduce mutations into the AYVV CP in pEAQ-HT-D1-AYVVCP at residues 41 (R to A: KS125P and KS126P), at residue 48, (R to A: KS127P and KS128P), and at residue 59 (M to D: KS129P and KS130P); Supplementary Table 2. pEAQ-HT-D1-AYVVCP was used as the DNA template for each reaction as described in the GeneArt site-directed mutagenesis system (Invitrogen). The sequences of the wild-type and mutated clones were verified prior to transforming Agrobacterium tumefaciens strain LBA 4404 for transient expression in plants. The AYVV satellite DNA-α partial repeat clone, pBin AYVV 1/7, was pressure co-infiltrated with pEAQ-HT-D1-AYVVCP or its mutants as described previously32 (link).
Hesketh E.L., Saunders K., Fisher C., Potze J., Stanley J., Lomonossoff G.P, & Ranson N.A. (2018). The 3.3 Å structure of a plant geminivirus using cryo-EM. Nature Communications, 9, 2369.
+ Open protocol
+ Expand
4

Purification and Mutagenesis of EPRS Linker

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Recombinant wild-type and Ser-to-Ala (S886A and S999A) mutant His-tagged linker proteins spanning Pro683 to Asn1023 of human EPRS were expressed and purified as described7 (link),8 (link). Recombinant active S6K132 (link) and RSK1–3 were from Cell Signaling; Akt1, and Akt2 were from EMD Millipore. Mouse EPRS domains ERS (Met1 (link) to Gln682), linker (Pro683 to Asn1023), and PRS (Leu1024 to Tyr1512) were cloned into pcDNA3 vector with an N-terminus Flag tag using full-length mouse EPRS cDNA (Origene) as template. Flag-tagged mouse wild-type linker and linker with Ser999-to-Ala (S999A) and Ser999-to-Asp (S999D) mutations were generated as described33 (link). Full-length human S6K1 cDNA in pCMV6-Entry vector was purchased from Origene and recloned, deleting the 23-amino acid N-terminus nuclear localization signal, and adding an in-frame upstream 6-His tag and a downstream Myc tag in pcDNA3. Specific Thr389-to-Ala (T389A) and Thr389-to-Glu (T389E) mutations were introduced using primers with the desired mutation and GENEART Site-Directed Mutagenesis System (Invitrogen).
Arif A., Terenzi F., Potdar A.A., Jia J., Sacks J., China A., Halawani D., Vasu K., Li X., Brown J.M., Chen J., Kozma S.C., Thomas G, & Fox P.L. (2017). EPRS is a critical mTORC1-S6K1 effector that influences adiposity in mice. Nature, 542(7641), 357-361.
+ Open protocol
+ Expand
5

Site-Directed Mutagenesis of Key Signaling Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols
Site-directed mutagenesis was conducted using GENEART® Site-Directed mutagenesis system according to the manufacturer’s instruction (Invitrogen) with NFκB2 (GeneCopoeia, Cat. EX-Z4293-Lv154), TLR2 (GeneCopoeia, Cat. EX-Q0161-Lv122), and mTOR (Addgene, Cat. 26603) expression vector. The primer sequences used for the site-directed mutagenesis are in the Supplemental Table 11.
Kim D., Park G., Huuhtanen J., Lundgren S., Khajuria R.K., Hurtado A.M., Muñoz-Calleja C., Cardeñoso L., Gómez-García de Soria V., Chen-Liang T.H., Eldfors S., Ellonen P., Hannula S., Kankainen M., Bruck O., Kreutzman A., Salmenniemi U., Lönnberg T., Jerez A., Itälä-Remes M., Myllymäki M., Keränen M.A, & Mustjoki S. (2020). Somatic mTOR mutation in clonally expanded T lymphocytes associated with chronic graft versus host disease. Nature Communications, 11, 2246.
+ Open protocol
+ Expand
6

Constructing Δ1D2A-GLuc/SGLuc Chimeras

Check if the same lab product or an alternative is used in the 5 most similar protocols
To construct Δ1D2A-GLuc/SGLuc Δ1M chimeras, pTarget Δ1D2A-GLuc and pTarget Δ1D2A-SGLuc template were used for site-directed mutagenesis using the GENEART Site-Directed Mutagenesis System (Invitrogen) with primers No Met GLuc-MF and No Met GLuc-MR (Table 1). Sequencing confirmation used primers T7 and Seq-R (Table 1), as described above.
Puckette M., Burrage T., Neilan J.G, & Rasmussen M. (2017). Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression. BMC Biotechnology, 17, 52.
+ Open protocol
+ Expand
7

Purification and Mutagenesis of EPRS Linker

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Recombinant wild-type and Ser-to-Ala (S886A and S999A) mutant His-tagged linker proteins spanning Pro683 to Asn1023 of human EPRS were expressed and purified as described7 (link),8 (link). Recombinant active S6K132 (link) and RSK1–3 were from Cell Signaling; Akt1, and Akt2 were from EMD Millipore. Mouse EPRS domains ERS (Met1 (link) to Gln682), linker (Pro683 to Asn1023), and PRS (Leu1024 to Tyr1512) were cloned into pcDNA3 vector with an N-terminus Flag tag using full-length mouse EPRS cDNA (Origene) as template. Flag-tagged mouse wild-type linker and linker with Ser999-to-Ala (S999A) and Ser999-to-Asp (S999D) mutations were generated as described33 (link). Full-length human S6K1 cDNA in pCMV6-Entry vector was purchased from Origene and recloned, deleting the 23-amino acid N-terminus nuclear localization signal, and adding an in-frame upstream 6-His tag and a downstream Myc tag in pcDNA3. Specific Thr389-to-Ala (T389A) and Thr389-to-Glu (T389E) mutations were introduced using primers with the desired mutation and GENEART Site-Directed Mutagenesis System (Invitrogen).
Arif A., Terenzi F., Potdar A.A., Jia J., Sacks J., China A., Halawani D., Vasu K., Li X., Brown J.M., Chen J., Kozma S.C., Thomas G, & Fox P.L. (2017). EPRS is a critical mTORC1-S6K1 effector that influences adiposity in mice. Nature, 542(7641), 357-361.
+ Open protocol
+ Expand
8

Transfection and Transcriptional Activation Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
U87MG cells or PFAs were seeded in 6, 12 or 24 well plates at a density designed to yield 90% confluency within 24 hours. Cells were transfected using Transfectin (Bio-Rad) or Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. pUC19 plasmid was used to normalize total transfection input DNA across all conditions. pmaxGFP plasmid (Lonza) was included to monitor transfection efficiency by fluorescence microscopy. M50 Super 8x TOPFlash β-catenin plasmid [77 (link)] (a gift from Randall Moon (Addgene plasmid # 12456; http://n2t.net/addgene:12456; RRID:Addgene_12456) and the Cignal TCF/LEF β-catenin plasmid (Qiagen #336841) are luciferase reporter constructs under the control of β-catenin-mediated transcriptional activation. Flag-Tat86WT and Flag-Tat101WT are HIV-1 Tat expression vectors that have been previously described [78 (link), 79 (link)]. Flag-Tat101WT was used to generate Tat mutants, Tat101C31R, Tat101W11F, and Tat101W11Y utilizing the GeneArt Site-Directed Mutagenesis System (Invitrogen).
Chen K., Phan T., Lin A., Sardo L., Mele A.R., Nonnemacher M.R, & Klase Z. (2020). Morphine exposure exacerbates HIV-1 Tat driven changes to neuroinflammatory factors in cultured astrocytes. PLoS ONE, 15(3), e0230563.
+ Open protocol
+ Expand
9

Construction of pEGFP-FAK Mutants

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
GeneArt Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA) was used for construction of pEGFP-FAK-H58A/E. Mutagenesis primers for H58A were the following: forward, 5′-C AGT ATT ATC AGG GCA GGA GAT GCT ACT GAT G and reverse, 5′-C ATC AGT AGC ATC TCC TGC CCT GAT AAT ACT G; for H58E: forward, 5′-C AGT ATT ATC AGG GAG GGA GAT GCT ACT GAT G and reverse, 5′-C ATC AGT AGC ATC TCC CTC CCT GAT AAT ACT G (the underlined triplets are those that are mutated). The mutation was verified by Sanger sequencing. Cells were transfected with the constructs by using Lipofectamine 3000 Transfection Reagent. G418 selection, fluorescence flow cytometry sorting, and colony expansion were performed as previously described.1 (link)
Zhang J., Gelman I.H., Katsuta E., Liang Y., Wang X., Li J., Qu J., Yan L., Takabe K, & Hochwald S.N. (2019). Glucose Drives Growth Factor–Independent Esophageal Cancer Proliferation via Phosphohistidine–Focal Adhesion Kinase Signaling. Cellular and Molecular Gastroenterology and Hepatology, 8(1), 37-60.
+ Open protocol
+ Expand
10

Mutagenesis of Rat 5-HT1B Receptor

Check if the same lab product or an alternative is used in the 5 most similar protocols
Point mutations were introduced into the wild-type rat 5-HT1B receptor sequence using the GeneArt Site-Directed Mutagenesis System (Invitrogen) according to manufacturer’s instructions with 20 ng starting DNA per reaction (Supplemental Table 2). Resulting clones were sequenced through their entire coding sequence to verify the successful introduction of the desired mutation and to confirm that no additional mutations had been introduced inadvertently.
Liu Y., Gibson A.W., Levinstein M.R., Lesiak A.J., Ong S.E, & Neumaier J.F. (2019). 5-HT1B RECEPTOR-MEDIATED ACTIVATION OF ERK1/2 REQUIRES BOTH Gαi/o AND β-ARRESTIN PROTEINS. ACS chemical neuroscience, 10(7), 3143-3153.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!