Q5 site directed mutagenesis kit

Manufactured by New England Biolabs

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The Q5 Site-Directed Mutagenesis Kit is a laboratory tool designed for introducing precise mutations into DNA sequences. It provides a streamlined workflow for generating site-specific changes in plasmid or linear DNA templates.

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Q5 site-directed mutagenesis (88 citations) Q5 mutagenesis kit (72 citations) Q5 mutagenesis (18 citations) Q5 kit (13 citations) Site-directed mutagenesis (8 citations) Q5 site Mutagenesis Kit (5 citations)

1 720 protocols using q5 site directed mutagenesis kit

PINK1 Knockout HeLa Cell Protocol

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HeLa PINK1^−/− cells were a gift from M. Lazarou (Monash University) and were authenticated at the Garvan Molecular Genetics facility using short tandem repeat profiling. Cells were cultured in DMEM supplemented with 10% (v/v) FBS (Gibco or Sigma-Aldrich), penicillin–streptomycin, and maintained at 37 °C and 5% CO₂. Cells were also screened routinely for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza). To limit the level of HsPINK1 overexpression, the pcDNA5/FRT/TO plasmid was modified using the Q5 site-directed mutagenesis kit (NEB) to generate a 539 bp deletion in the CMV promoter (CMVd3)⁵⁸, hereafter referred to as pcDNA5^d3. The full-length, WT HsPINK1 sequence was inserted into the BamHI site of pcDNA5^d3 using In-Fusion Cloning (Takara Bio) and used for transient transfections. For stable HsPINK1 expression, the HsPINK1 sequence was inserted into the BamHI and NheI of the lentiviral pFU PGK Hygro (pFUH) plasmid using InFusion Cloning. All of the HsPINK1 mutants were generated using the Q5 site-directed mutagenesis kit (NEB).

Gan Z.Y., Callegari S., Cobbold S.A., Cotton T.R., Mlodzianoski M.J., Schubert A.F., Geoghegan N.D., Rogers K.L., Leis A., Dewson G., Glukhova A, & Komander D. (2021). Activation mechanism of PINK1. Nature, 602(7896), 328-335.

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Generating ITGB4 Plasmid with Genetic Variants

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A TrueORF gold ITGB4 plasmid with a c-terminal Myc-DDK tag was obtained from Origene (RC220541, Origene Technologies, Rockville, MD, USA). The ITGB4 genetic variants rs147480547 allele A and rs145976111 allele T were introduced into the plasmid using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs) and following the manufacturer's protocol. A plasmid containing both of the genetic variants was also constructed using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, UK).

O'Brien N.L., Fiorentino A., Curtis D., Rayner C., Petrosellini C., Al Eissa M., Bass N.J., McQuillin A, & Sharp S.I. (2018). Rare variant analysis in multiply affected families, association studies and functional analysis suggest a role for the ITGΒ4 gene in schizophrenia and bipolar disorder. Schizophrenia Research, 199, 181-188.

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Plasmid Constructs for Enterovirus Research

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p3A(CVB3)-myc (27 (link)), pEGFP-3A(RV-2), and pEGFP-3A(RV-14) were described previously (19 (link)). p3A(EV-A71)-myc, p3A(PV1)-myc, pEGFP-3A(EV-D68), and pEGFP-3A(RV-C15) were prepared by cloning cDNA encoding EV-A71 and PV1 3A into p3A(CVB3)-myc vectors from which CVB3 3A was excised using restriction enzyme sites SalI and BamHI, and EV-D68 and RV-C15 3A into pEGFP vectors using restriction enzyme sites BglII and BamHI. pEGFP-GalT was a gift from Jennifer Lippincott-Schwartz (Addgene plasmid 11929). pEGFP-ACBD3 was a gift from Carolyn E. Machamer (Johns Hopkins University, USA). pEGFP-ACBD3-FQ and pEGFP-ACBD3-mut1/mut2/mut3 were generated by using a Q5 site-directed mutagenesis kit (New England BioLabs). pCDNA3-FLAG-PI4KB(wt) was a gift from Tamas Balla (NIH, USA). pCDNA3-FLAG-PI4KB(D⁶⁷¹A) (kinase activity dead [KD] mutant), pCDNA3-FLAG-PI4KB(I⁴³A), and pCDNA3-FLAG-PI4KB(D⁴⁴A) were generated by using a Q5 site-directed mutagenesis kit (New England BioLabs).

Lyoo H., van der Schaar H.M., Dorobantu C.M., Rabouw H.H., Strating J.R, & van Kuppeveld F.J. (2019). ACBD3 Is an Essential Pan-enterovirus Host Factor That Mediates the Interaction between Viral 3A Protein and Cellular Protein PI4KB. mBio, 10(1), e02742-18.

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Generating Mutant BKPyV VP1 Plasmids

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The VP2 and VP3 expression plasmids ph2b and ph3b (#32109 and #32110) were obtained from Addgene (Cambridge, MA, USA). VP1 expression plasmids for genotypes Ia, Ib2 and IVc2 were kindly provided by Dr Christopher Buck, National Cancer Institute (NCI), Bethesda, MD. The plasmid pEGFP-N1 (Clontech) was used as the reporter gene. Mutations were introduced into the relevant VP1 plasmids by site-directed mutagenesis using the Q5 Site-directed mutagenesis kit (New England Biolabs, Evry, France). Primer pairs used for mutagenesis were selected using the NEBasechanger tool. After each mutagenesis reaction, miniprep DNA from four colonies was screened by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany) with the EF1a-F primer. The full-length VP1 sequence of clones incorporating the desired mutations was confirmed by sequencing with the WPRE-R primer. The BKPyV-MM genome cloned into the pBR322 vector (pBKV 35-1, ATCC 45026) was obtained from the ATCC, and VP1 mutations were introduced using the Q5 Site-directed mutagenesis kit (New England Biolabs).

McIlroy D., Hönemann M., Nguyen N.K., Barbier P., Peltier C., Rodallec A., Halary F., Przyrowski E., Liebert U., Hourmant M, & Bressollette-Bodin C. (2020). Persistent BK Polyomavirus Viruria Is Associated with Accumulation of VP1 Mutations and Neutralization Escape. Viruses, 12(8), 824.

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Recombinant Expression of S. degradans β-Mannanase

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The genes encoding the S. degradans β-mannanase SdGH5_8-CBM10x3 (GenBank accession no.: ABD79918) without the predicted signal peptide (amino acids 1–21) and the three CBM10s Sd_GH5CBM10x3 fused with GFP, and GFP (AGT98536) were purchased (GeneArt; Thermo Fisher Scientific), and cloned into the pET28a(+) vector using the XhoI and NheI restriction sites resulting in an N-terminal cleavable His tag. The three C-terminally truncated forms SdGH5_8-CBM10x2, SdGH5_8-CBM10x1, and SdGH5_8 (Fig. 2) were constructed by introducing stop codons using Q5 Site-Directed Mutagenesis Kit (New England Biolabs), pET28a-SdGH5_8-CBM10x3 as template and mutagenesis primers (Table S1) designed according to the manufacturers' instructions. Each of the three Sd_GH5CBM10s were obtained as Sd_GH5CBM10 to 1_GFP, Sd_GH5CBM10 to 2_GFP, and Sd_GH5CBM10-3_GFP by mutagenesis using Q5 Site-Directed Mutagenesis Kit (New England Biolabs), pET28a-Sd_GH5CBM10x3_GFP as template and mutagenesis primers. To get Sd_GH5CBM10-1_GFP, both Sd_GH5CBM10-2 and Sd_GH5CBM10-3 were deleted in one step. A one-step deletion of Sd_GH5CBM10-1 and Sd_GH5CBM10-2 was done to get Sd_GH5CBM10-3-GFP. Finally, Sd_GH5CBM10-2_GFP was obtained in two steps; first deleting CBM10-1 and then CBM10-3. The gene constructs were confirmed by sequencing.

Møller M.S., El Bouaballati S., Henrissat B, & Svensson B. (2021). Functional diversity of three tandem C-terminal carbohydrate-binding modules of a β-mannanase. The Journal of Biological Chemistry, 296, 100638.

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Cloning and Mutagenesis of PAR-1 and MEX-5 Constructs

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Table S3 lists all plasmids used in this study. A Q5 site-directed mutagenesis kit (New England Biolabs) was used to integrate the 6xHis coding sequence at the N-terminus of the maltose-binding protein (MBP) coding sequence in vector pMAL-C5E (New England Biolabs), to generate pAF9. The 6xHis::MBP::PAR-1 and 6xHis::MBP::PAR-1(ΔKA1) expression vectors were constructed by cloning par-1 and par-1(ΔKA1) open reading frames (ORFs) into pAF9. The 6xHis::MBP::MEX-5(452-460) expression vector was constructed by cloning mex-5 ORFs into pAF9 using a Gibson assembly cloning kit (New England Biolabs). The 6xHis::KA1 expression vector (pAF10) was constructed by cloning par-1(1089-1192) ORF into pET28a (Novagen) using a Gibson assembly cloning kit (New England Biolabs). A Q5 site-directed mutagenesis kit (New England Biolabs) was used to generate the 6xHis::KA1(KRSS) expression vector (pAF11) (Tables S3,S4).

Folkmann A.W, & Seydoux G. (2019). Spatial regulation of the polarity kinase PAR-1 by parallel inhibitory mechanisms. Development (Cambridge, England), 146(6), dev171116.

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Recombinant Protein Expression and Mutagenesis

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Wild type (WT) HU αA-crystallin of 173 amino acids was overexpressed using a pET23d expression vector (EMD Millipore, Novagen,Billerica, MA, U.S.A.). Mutant HU C142S αA-crystallin, with a substitution of one cysteine at residue 142, was created with primers designed by NEBaseChanger™, online software for primer design, and with a Q5 Site-Directed Mutagenesis Kit from New England Biolabs (NEB, Ipswich, MA, U.S.A.). The gene for GP αA^ins-crystallin, containing an insert of 23 amino acids in the region from residue 63 of WT GP αA-crystallin, was synthesized by Genscript (Piscataway, NJ, U.S.A.) and cloned into a pET28a expression vector between the NcoI and XhoI restriction sites. A plasmid system for expression of normal WT GP αA-crystallin of 173 amino acids was created by deletion of the 69 base pair insert in the GP αA^ins- crystallin gene with primers designed with NEBaseChanger™, and with use of a Q5 Site- Directed Mutagenesis Kit from NEB. An S142C mutant of GP αA-crystallin, containing two cysteines rather than the one present in the WT protein, was created in a similar manner. All plasmid constructs used were sequenced to confirm identity (Supplemental Fig. 1).

Kumarasamy A., Jeyarajan S., Cheon J., Premceski A., Seidel E., Kimler V.A, & Giblin F.J. (2018). Peptide-induced formation of protein aggregates and amyloid fibrils in human and guinea pig αA-crystallins under physiological conditions of temperature and pH. Experimental eye research, 179, 193-205.

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Site-Directed Mutagenesis of BCL11A

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All constructs incorporating the single nucleotide variants of interest were created using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturer’s recommendations. HA-tags were introduced to the C-terminus of the BCL11A WT and Mut-4 constructs via Q5 Site-Directed Mutagenesis Kit (New England Biolabs) following the manufacturers recommendations for insertions. A glycine-serine linker (G-S-S-G) was added at the C-terminal end of the HA-tag. Primers used for constructs are listed in S1 Table.

Shen Y., Li R., Teichert K., Montbleau K.E., Verboon J.M., Voit R.A, & Sankaran V.G. (2021). Pathogenic BCL11A variants provide insights into the mechanisms of human fetal hemoglobin silencing. PLoS Genetics, 17(10), e1009835.

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Molecular Cloning of hnRNPDL Isoforms

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hnRNPDL isoform 1 sequence was amplified by PCR from cDNA extract of U2OS cells and assembled to pEGFP-C3 HindIII and BamHI digested plasmid using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). Similar to bacterial molecular cloning, pEGFP-C3-DL1 plasmid was used as a template for isoform 2 (DL2) construct using the Q5 site directed mutagenesis kit (New England Biolabs). hnRNPDL isoform 3 (DL3) was obtained using pEGFP-C3-DL2 plasmid as a template and the Q5 site directed mutagenesis kit (New England Biolabs). The final vectors were transformed into One Shot TOP10 chemically competent E. coli cells (Thermo Fisher Scientific).

Batlle C., Yang P., Coughlin M., Messing J., Pesarrodona M., Szulc E., Salvatella X., Kim H.J., Taylor J.P, & Ventura S. (2020). hnRNPDL Phase Separation Is Regulated by Alternative Splicing and Disease-Causing Mutations Accelerate Its Aggregation. Cell Reports, 30(4), 1117-1128.e5.

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Cloning and Transgenesis of PlexB

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The sequence encoding wild-type PlexB (Joo et al., 2013 (link)) was amplified by Q5 hot-start high-fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) and assembled into a pUAST-attB vector (Li et al., 2017 (link)) by NEBuilder HiFi DNA assembly master mix (New England Biolabs, Ipswich, MA, USA). A V5 tag was inserted before the stop codon by Q5 site-directed mutagenesis kit (New England Biolabs, Ipswich, MA, USA). Afterwards, deletions and point mutations were introduced by Q5 site-directed mutagenesis kit (New England Biolabs, Ipswich, MA, USA). All constructs were transformed into NEB stable competent E. coli (New England Biolabs, Ipswich, MA, USA), extracted by QIAprep spin miniprep kit (QIAGEN, Hilden, Germany), and verified by full-length sequencing (Elim Biopharmaceuticals, Hayward, CA, USA). Constructs were then injected into vas-int.Dm;;ZH-attP-86Fb embryos (Bischof et al., 2007 (link)). White+ progenies were individually balanced by TM3 or TM6B, with the vas-int.Dm transgene removed.

Guajardo R., Luginbuhl D.J., Han S., Luo L, & Li J. (2019). Functional divergence of Plexin B structural motifs in distinct steps of Drosophila olfactory circuit assembly. eLife, 8, e48594.

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