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Stratagene quikchange 2 xl site directed mutagenesis kit

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The Stratagene QuikChange II XL Site-Directed Mutagenesis Kit is a laboratory equipment product designed for introducing site-specific mutations into double-stranded plasmid DNA. The kit provides the necessary reagents and protocols to perform the mutagenesis process.

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Lab products found in correlation

X tremegene hp, by Roche (2 mentions) Puromycin, by Merck Group (1 mentions) Pfuultra 2 fusion hs dna polymerase, by Agilent Technologies (1 mentions) Gateway technology, by Thermo Fisher Scientific (1 mentions)

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QuikChange II Site-Directed Mutagenesis Kit (865 citations) QuikChange II XL Site-Directed Mutagenesis Kit (647 citations) QuikChange site-directed mutagenesis (131 citations) QuikChange II Site-Directed Mutagenesis (24 citations) Quikchange II-XL Site Directed Mutagenesis (9 citations) Quikchange XL site-directed mutagenesis (3 citations) QuikChange II site-directed (3 citations) Stratagene Quikchange II site-directed mutagenesis kit (2 citations) XL II Site-Directed Mutagenesis Kit (2 citations) QuikChange II Directed Mutagenesis Kit (2 citations)

5 protocols using stratagene quikchange 2 xl site directed mutagenesis kit

1

Exon-trapping Analysis of PKHD1 Variants

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For exon-trapping minigene analysis, constructs containing mouse Pkhd1 exons 6, 7, and either, 45 (negative control), 49, 51, or 52 were generated in the exon-trapping vector pSPL3 [21 (link)]. For evaluating the R760H missense variant, either normal or mutant PKHD1 exons 21, 22, and 23 were incorporated into the vector. The genomic DNA included at least 200 bp of intronic sequence flanking each exon. Total RNA was purified from COS cells 48 h post-transfection. cDNA was generated by reverse transcription using 4 µg of total RNA. Transcripts were amplified by PCR and analyzed by sequencing. The vector based primer sets used for PCR reaction were “a” (5-ATCTCA GTGGTATTTGTGAGC-3) and “b” (5-TCTGAGTCACCT GGACAACC-3). Site-directed mutagenesis was performed using Stratagene QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions.
Boddu R., Yang C., O’Connor A.K., Hendrickson R.C., Boone B., Cui X., Garcia-Gonzalez M., Igarashi P., Onuchic L.F., Germino G.G, & Guay-Woodford L.M. (2014). Intragenic motifs regulate the transcriptional complexity of Pkhd1/PKHD1. Journal of molecular medicine (Berlin, Germany), 92(10), 1045-1056.
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2

Overexpression of SNPH, SOD2, and PrxIII

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True-ORF pCMV6-Entry-myc-Flag encoding SNPH (cat# RC207749), SOD2 (cat# RC202330) and PrxIII (cat# RC205080) were from Origene. SNPH mutants lacking the microtubule binding domain (ΔMTB, Δ86-159 aa), LC8-binding domain (ΔLC8, Δ311-317 aa) or kinesin-binding domain (ΔKBD, Δ337-422 aa) were generated with the Stratagene QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies), and confirmed by DNA sequencing. Cells were transfected with 2 μg of pcDNA plus 4 μl X-treme gene HP (Roche) for 24 h in complete medium, washed and subject to the indicated treatments. Cells stably expressing full length SNPH, SNPH mutants or an empty vector were generated by transfection of plasmids, followed by a 2-week selection in the presence of 1 mg ml−1 G418 (Geneticin).
Caino M.C., Seo J.H., Aguinaldo A., Wait E., Bryant K.G., Kossenkov A.V., Hayden J.E., Vaira V., Morotti A., Ferrero S., Bosari S., Gabrilovich D.I., Languino L.R., Cohen A.R, & Altieri D.C. (2016). A neuronal network of mitochondrial dynamics regulates metastasis. Nature Communications, 7, 13730.
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3

Mouse PC1 Expression in HEK293T and HeLa Cells

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HEK293T and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with L-glutamine, penicillin-streptomycin, and 10% fetal bovine serum at 37 °C and 5% CO2. HEK293T cells stably expressing Flag-tagged full length mouse PC1 was a generous gift of Dr. J. Yang (Columbia University, NY) and cultured under above-described medium supplemented with 2 µg/ml of puromycin (Sigma-Aldrich Canada)46 (link). pcDNA3 plasmids encoding the N-terminal GFP-tagged PC1 C-terminus (PC1C, aa 4104-4302), last 5 TMs plus PC1C (PC1-5TMC, aa 3895–4302), and last TM plus PC1C (PC1-1TMC, aa 4084–4302) were constructed by site-directed mutagenesis using Stratagene QuikChange® II XL Site-Directed Mutagenesis Kit (Agilent Technologies Canada Inc., Mississauga, ON). HEK293T cells were grown to ~70% confluency prior to transfection using Lipofectamine 2000 (Invitrogen Canada Inc., Burlington, ON). All plasmid construction and cDNA sequences were verified by sequencing.
Tang Y., Wang Z., Yang J., Zheng W., Chen D., Wu G., Sandford R., Tang J, & Chen X.Z. (2017). Polycystin-1 inhibits eIF2α phosphorylation and cell apoptosis through a PKR-eIF2α pathway. Scientific Reports, 7, 11493.
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4

Parkin Mutants and In Vitro Ubiquitination

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A cDNA for WT Parkin was from GeneCopoeia. E3 ligase loss-of-function Parkin Ser65Ala (S65A) and Cys431Ser (C431S) mutants were generated using PfuUltra II fusion HS DNA Polymerase (Agilent Technologies). Ub-defective K2 mutant Lys581Ala (K581A), Lys 582Ala (K582A), or DM Lys581Ala/Lys582Ala (K581A/K582A) were generated using Stratagene QuikChange II XL Site-Directed Mutagenesis kit (Agilent Technologies) and confirmed by DNA sequencing. Protein expression was assessed by Western blotting and visualized by chemiluminescence (26 ). In vitro Ub studies were carried out as described (37 (link)).
Yeon M., Bertolini I., Agarwal E., Ghosh J.C., Tang H.Y., Speicher D.W., Keeney F., Sossey-Alaoui K., Pluskota E., Bialkowska K., Plow E.F., Languino L.R., Skordalakes E., Caino M.C, & Altieri D.C. (2023). Parkin ubiquitination of Kindlin-2 enables mitochondria-associated metastasis suppression. The Journal of Biological Chemistry, 299(6), 104774.
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5

Characterization of SNPH Ubiquitination

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The following recombinant proteins were used: UBE1 (E1, UBPBio, cat.n. #B1100), His-UBE2D1 (E2, UBPBio, cat.n. C1400), His-UBE2D3 (E2, UBPBio, cat.n. C1600), His-UBE2E3 (E2, UBPBio, cat.n. C2000), ubiquitin (UBPBio, cat.n. E1100). Recombinant CHIP (Stub1) was purchased from Boston Biochem. A SNPH mutant lacking the kinesin-binding domain (ΔKBD, Δ337–422 aa) has been characterized previously (16 (link)). Ubiquitination-defective SNPH mutants K111R, K153R, K175R, K295R and K415R were generated using Stratagene QuikChange II XL Site-Directed Mutagenesis kit (Agilent Technologies), and confirmed by DNA sequencing. Cells were transfected with 2 μg of pDNA plus 4 μl X-treme gene HP (Roche) for 24 h in complete medium, washed and processed for individual experiments. Adenoviral vectors expressing LacZ, wild type (WT) SNPH, or the various ubiquitination-defective SNPH mutants were produced using Gateway technology (Thermo Fisher Scientific) with pDONR221 vector and recombination into the adenovirus expression vector pAd/CMV/V5-DEST. After digestion with PacI, the individual constructs were transfected in 293A cells for production of adenoviruses after 7 d.
Seo J.H., Agarwal E., Bryant K.G., Caino M.C., Kim E.T., Kossenkov A.V., Tang H.Y., Languino L.R., Gabrilovich D.I., Cohen A.R., Speicher D.W, & Altieri D.C. (2018). SYNTAPHILIN UBIQUITINATION REGULATES MITOCHONDRIAL DYNAMICS AND TUMOR CELL MOVEMENTS. Cancer research, 78(15), 4215-4228.
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