Cmyc-R11.1.6, cmyc-YW1, HA-K-Ras G12D, HA-K-Ras WT, EGFP-R11.1.6, EGFP-YW1, and mApple-K-Ras G12D were cloned into the gWIZ vector (Genlantis) for mammalian expression using In-Fusion Cloning (Clontech) according to the manufacturer’s instructions. The sequence for R11.1.6 was PCR amplified from the yeast display vector (pCTCON2), digested with BsaI and XbaI, and cloned into BsaI digested pE-SUMO-vector (LifeSensors) for bacterial protein expression. To make the scrambled YW1 control and R11.1.6 binding interface alanine point mutants, the QuikChange site-directed mutagenesis method (Agilent) was used according to the manufacturer’s instructions. Transient transfections into HEK 293T cells were carried out using calcium phosphate. Briefly, DNA diluted in water was added to 2 M CaCl2, to which 2x HBS was added dropwise. The transfection mixture was added to plated cells and incubated for 8 hours, after which the transfection medium was replaced with complete medium. Unless indicated otherwise, the ratio of DNA transfected for K-Ras constructs to R11.1.6/YW1 constructs was 1:4.
Quikchange site directed mutagenesis method
Manufactured by Agilent Technologies
Sourced in United States
The QuikChange site-directed mutagenesis method is a laboratory technique used to introduce specific mutations into double-stranded plasmid DNA. The method involves the use of a pair of synthetic oligonucleotide primers that contain the desired mutation and can anneal to opposite strands of the plasmid. The primers are then extended using a DNA polymerase, resulting in the incorporation of the mutation into the plasmid.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Pflag cmv 5a, by Merck Group (2 mentions)
In fusion cloning, by Takara Bio (1 mentions)
Gwiz vector, by Genlantis (1 mentions)
Pe sumo vector, by LifeSensors (1 mentions)
Cdna clone, by Source Bioscience (1 mentions)
Pcdna3.1 vector, by Thermo Fisher Scientific (1 mentions)
Pdsred er, by Takara Bio (1 mentions)
Pdsred monomer golgi, by Takara Bio (1 mentions)
Pac5.1 v5 his vector, by Thermo Fisher Scientific (1 mentions)
Pentr d topo, by Thermo Fisher Scientific (1 mentions)
Plasmid midi kit, by Qiagen (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Flag antibody, by Cell Signaling Technology (1 mentions)
Mab1501, by Merck Group (1 mentions)
E coli bl21 de3 competent cells, by New England Biolabs (1 mentions)
One shot top10 chemically competent e coli, by Thermo Fisher Scientific (1 mentions)
Pfuultra 2 fusion hotstart dna polymerase, by Agilent Technologies (1 mentions)
Bac to bac system, by Thermo Fisher Scientific (1 mentions)
25 protocols using quikchange site directed mutagenesis method
1
Mammalian Expression and Bacterial Purification of Engineered Proteins
2
Characterization of TRPM2 Phosphorylation Sites
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Mouse TRPM2 cDNAs in the pCIneo expression vector were generously provided by Dr Mori (Kyoto Univ., Kyoto, Japan). Mouse TRPM2 or hemagglutinin (HA)‐tagged mouse TRPM2 cDNAs were cloned into pcDNA3.1(+) by PCR‐based cloning. Vectors containing the full‐length mutants were constructed by single amino acid substitutions using a modified QuikChange Site‐Directed Mutagenesis method (Agilent Technologies, Inc., Santa Clara, CA, USA). Candidate Ser/Thr residues involved in phosphorylation by PKC were predicted by NetPhos‐3.1(https://services.healthtech.dtu.dk/service.php?NetPhos‐3.1 ). Synthetic oligonucleotide primers containing specific mutations are shown in Table 1 . Expression vectors containing alanine mutations in full‐length TRPM2 cDNA were reconstructed in the pcDNA3.1(+) expression vector. The entire sequence including the desired alanine substitutions in the mutants was confirmed by DNA sequencing.
3
Site-Directed Mutagenesis and Transformation in Vibrio alginolyticus
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Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis method, as described by Agilent Technologies (Santa Clara, USA). Transformation of V. alginolyticus by plasmids pHFAB or pYA303 was carried out by electroporation42 . Transformation of V. alginolyticus by plasmid pNT1 was carried out by conjugational transfer from E. coli S17-143 (link).
4
Cloning and Mutagenesis of Mouse Slc26a9
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DNA encoding the open reading frame (ORF) for mouse Slc26a9 (GenBank accession: BC160193) was PCR-amplified from a cDNA clone (Source BioScience) and shuttled into a pcDNA 3.1 vector (Invitrogen) which was modified to be compatible with FX cloning technology (Geertsma and Dutzler, 2011 (link)). Unless stated otherwise, all expression constructs also encoded a C-terminal Rhinovirus 3C protease cleavage site followed by venus YFP (vYFP), a myc epitope tag and a streptavidin binding peptide (SBP), giving the general construct scheme ORF-3C-vYFP-myc-SBP. Assembly of the Slc26a9T dual-truncation construct entailed removal of the STAS IVS region, via replacement of residues Pro558–Val660 with a Gly-Ser linker, and deletion of C-terminal residues Pro745–Leu790, akin to a described procedure for the isolated STAS domain from rat Prestin (Pasqualetto et al., 2010 (link)). Further deletion of N-terminal residues Met1–Ala30 resulted in the construct Slc26a9(Δ1-30)T. Mutations Q88A, Q88E, F92A, T127A, F128A, L391A, S392A, R205E, K221E, K270E, K431E, and K441E were introduced into Slc26a9T using the QuikChange site-directed mutagenesis method (Agilent).
5
GLT8D1 Expression Construct Generation
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A full-length coding region of GLT8D1 was cloned into pFLAG-CMV-5a (Sigma-Aldrich, St. Louis, MO) to generate the wild-type (WT) GLT8D1 expression construct. The 2 GLT8D1 variations, p.I290M (c.870C>G) and p.R92C (c.274 G>A), were separately introduced into the WT expression plasmids by using the QuikChange Site-Directed Mutagenesis method (Agilent, Santa Clara, CA). The endoplasmic reticulum (ER) marker pDsRed-ER and the Golgi marker pDsRed-Monomer-Golgi were purchased from Clontech (Mountain View, CA). HEK293T cells were maintained in Dulbecco modified eagle medium supplemented with 10% FBS. Transient transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific).
6
Constructing SKIP Truncation Mutants
7
Cloning and tagging of dIPIP and IPIP27A
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The CG12393 cDNA sequence encoding dIPIP (DGRC clone SD10969) was amplified by PCR to include an N-terminal Myc tag and subcloned into the pAc5.1-V5-His vector (Invitrogen). dIPIP C-terminally tagged with mRuby was generated by cloning dIPIP cDNA into a modified pAc5.1-V5-His A vector (obtained from Richard Baines’ lab, Manchester). dIPIP cDNA was cloned into the pGEX-6P-1 vector to generate a GST fusion. Human Myc- tagged IPIP27A cDNA was cloned into the pAc5.1-V5-His vector. GFP- and mCh-dRab35 constructs were generated by PCR amplification of the ORF from cDNA (DGRC Clone LD21953, Vincent Archambault’s lab, Montreal), followed by cloning into pENTR-D-TOPO (Invitrogen) followed by recombination using LR Clonase into the pMT-ChW destination vector (Drosophila Gateway Vector Collection; T. Murphy, Carnegie Institution for Science, Washington, DC). Mutagenesis was performed using the Quikchange site-directed mutagenesis method (Agilent technologies). All constructs were verified by DNA sequencing (GATC Biotech).
8
Heterologous Expression of NaChBac and NavMs
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WT NaChBac cDNA in a modified pTracer-CMV2 expression vector was a gift from D. Ren (University of Pennsylvania, Philadelphia, PA), and WT NavMs cDNA in a modified pTracer-CMV2 expression vector was a gift from P. DeCaen (Northwestern University, Evanston, IL) and D. Clapham (Harvard University, Boston, MA). cDNA was amplified in bacterial culture and purified with the QIAGEN Plasmid Midi kit. To generate NaChBac T220A, a point mutation was introduced into the WT plasmid using the QuikChange site-directed mutagenesis method (Agilent).
HEK-293 (NaChBac) or HEK-293T (NavMs) cells were transiently transfected with cDNA using the Lipofectamine 2000 transfection reagent (Invitrogen) and seeded onto 12-mm circular glass coverslips 24 h before patch clamp recording. Standard protocols were followed for growth and maintenance of cells in culture.
HEK-293 (NaChBac) or HEK-293T (NavMs) cells were transiently transfected with cDNA using the Lipofectamine 2000 transfection reagent (Invitrogen) and seeded onto 12-mm circular glass coverslips 24 h before patch clamp recording. Standard protocols were followed for growth and maintenance of cells in culture.
9
Optimized PspR expression in E. coli
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The gene encoding PspR with codons optimized for Escherichia coli (E. coli) expression was synthesized by GenScript and cloned into NdeI–XhoI site of pET21a (+) vector (Novagen, Merck KGaA). The plasmid was transformed into E. coli C43 (DE3) strain (Lucigen). For mutagenesis, the QuikChange site-directed mutagenesis method (Agilent Technologies) was used according to a standard protocol. The sequences of the primers used in mutagenesis are listed in Table S2 .
10
Site-Directed Mutagenesis and Protein Expression
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Point mutations were made using the QuikChange site-directed mutagenesis method (Agilent). Baculovirus production and protein expression were performed by the Protein Expression Laboratory at the National Cancer Institute as detailed in Supplementary Notes .
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