Q5 mutagenesis kit

Manufactured by New England Biolabs

Sourced in United States

The Q5 mutagenesis kit is a specialized tool for introducing site-directed mutations in DNA sequences. It provides a simple and efficient method to generate desired genetic modifications in a wide range of applications.

Automatically generated - may contain errors

Lab products found in correlation

In fusion hd cloning kit, by Takara Bio (3 mentions) Lentiviral vector encoding flag tagged cpt1b, by OriGene (2 mentions) Pspax2, by Addgene (2 mentions) Pmd2 g, by Addgene (2 mentions) Lr clonase 2 reaction, by Thermo Fisher Scientific (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Pcdna3, by Thermo Fisher Scientific (1 mentions) Pcdna3, by GenScript (1 mentions) Sanger sequencing, by Eurofins (1 mentions) Pegfp n1 vector, by Takara Bio (1 mentions) Pcdna3 clover, by Addgene (1 mentions) Carbenicillin, by GoldBio (1 mentions) Digitonin, by Merck Group (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions) Triton x 100, by Merck Group (1 mentions) Egfp n3 vector, by Takara Bio (1 mentions) Pcmv ha myc plasmid, by Takara Bio (1 mentions) Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions) Primescript 1st strand cdna synthesis kit, by Takara Bio (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions)

72 protocols using q5 mutagenesis kit

CSNAP C-Terminal Truncation Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

The C-terminal ⁴²DFFNDFEDLFDDDDIQ⁵⁷ (C-CSNAP) sequence of CSNAP was generated in pHyg vector coding for full-length CSNAP fused to the N-terminus of Cer through a short Gly–Ser–Gly–Ser linker, by deleting ¹M-⁴¹A residues of CSNAP using Q5 mutagenesis kit (NEB). N- (-⁴²D, -⁴²DF) and C-terminal truncations (-⁵⁷Q, -⁵⁶IQ, -⁵⁵DIQ, and -⁵⁴DDIQ) were performed on phyg–C-CSNAP–Cer using the Q5 mutagenesis kit (NEB) (Table S3). HAP1 WT cells were transfected with 5 μg plasmid DNA using jetPRIME (Polyplus), and the culture medium was changed 4 h post-transfection. Stably expressing WT cell lines were prepared by applying hygromycin (0.4 mg/ml) selection for 7 d following transfection, then the pool of surviving cells were sorted on a FACSAriaIII cell sorter for Cer-positive cells.
Expression levels were monitored by loading 30 μg of each lysate on 12% Laemmli SDS–PAGE, transferred to PVDF membrane, and probed with anti-GFP antibody (ab290; Abcam).

Füzesi-Levi M.G., Ben-Nissan G., Listov D., Fridmann Sirkis Y., Hayouka Z., Fleishman S, & Sharon M. (2023). The C-terminal tail of CSNAP attenuates the CSN complex. Life Science Alliance, 6(10), e202201634.

+ Open protocol

+ Expand

Generation and Characterization of iCRISPR-Cas9 hiPSC Lines

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

The human iPS cell line 409B2 was used to create an iCRISPR–Cas9 nickase (Cas9n) and an iCRISPR–Cas9 line as described previously^{48 (link)}. The doxycycline-inducible Cas9-expressing cell line was generated by introducing two transcription activator-like effector nucleases (TALENs) targeting the AAVS1 locus, which has shown to be effective for sustained transgene expression, and two TALEN constructs with donor plasmids. One of the donor plasmids contained a constitutive reverse tetracycline transactivator (AAVS1-Neo-M2rtTA) and the other one contained a doxycycline-inducible expression cassette (Puro-Cas9). A D10A mutation was introduced by site-directed mutagenesis of the original Puro-Cas9 donor using the Q5 mutagenesis kit (New England Biolabs, E0554S) to generate the Cas9n. The cell lines used were tested for the proper expression of pluripotency markers SOX2, OCT4, TRA-1-60 and SSEA, quantitative PCR confirmed the doxycycline-inducible Cas9n and digital PCR was used to exclude off-target integration^{49 (link)}. Both cell lines showed normal karyotypes after generation, but the iCRISPR–Cas9 line acquired a common stem cell abnormality over time. A total of 55% percent of the cells showed a derivative chromosome 2 with a long arm of chromosome 1 (bands q11q44) attached to the long arm of one chromosome 2 (band q37).

Fleck J.S., Jansen S.M., Wollny D., Zenk F., Seimiya M., Jain A., Okamoto R., Santel M., He Z., Camp J.G, & Treutlein B. (2022). Inferring and perturbing cell fate regulomes in human brain organoids. Nature, 621(7978), 365-372.

+ Open protocol

+ Expand

Recombinant Expression of RORγ Isoforms

Check if the same lab product or an alternative is used in the 5 most similar protocols

RORγ2 was codon optimized (Genewiz) and subcloned into the pESUMO vector using XhoI and XbaI. The RORγ1 construct was generated by PCR insertion with the Q5 mutagenesis kit (New England Biolabs). Both constructs were transformed in BL21 (DE3) expression E. coli and selected on Luria broth agar plates containing 100 ug/mL ampicillin. 6–8 colonies were picked for primary culture in 250 mL of terrific broth and cultured for 16 h at 37°C. 15 mL of primary culture was diluted into 1 L of terrific broth supplemented with 30 μM ZnCl₂ and 50 ug/mL carbenicillin. The secondary culture was incubated at 37°C with shaking at 200 RPM until the optical density at 610 nm reached 0.5 after which the temperature was dropped to 16°C. protein expression was induced by addition of IPTG to 250 μM and the culture was incubated at 16°C for 8 h and then 4°C until harvest. The cells were harvested by centrifugation (4000 rcf for 10 minutes at 4°C), resuspended in ice cold NiNTA buffer 1 (50 mM HEPES pH 8.0 at 4°C, 500 mM NaCl, 10% glycerol, 25 mM imidazole) supplemented with 1X SigmaFast protease inhibitor cocktail, and pelleted again by centrifugation (4000 RCF for 10 minutes at 4°C). Harvested cells were flash frozen in liquid nitrogen and stored at −80°C until purification.

Strutzenberg T.S., Zhu Y., Novick S.J., Garcia-Ordonez R.D., Doebelin C., He Y., Chang M.R., Kamenecka T.M., Edwards D.P, & Griffin P.R. (2021). Conformational Changes of RORγ During Response Element Recognition and Coregulator Engagement. Journal of molecular biology, 433(22), 167258.

+ Open protocol

+ Expand

Generating Missense Mutations in ABCA4

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Missense mutations in ABCA4-1D4 were generated by PCR based site-directed mutagenesis as previously described^{21 ,22 (link)} Briefly, mutagenesis was performed using a cloning cassette encoding F213 (Afe1 restriction site) to R943 (Fse1 restriction site) in pcDNA3. PCR was performed with Q5 mutagenesis kit (New England Biolabs) with overlapping primers (Supplementary Table 2) as per manufacturer’s recommendations. Each amplified ABCA4 F213-R943 cDNA was sequenced before cloning back into pCEP4 plasmid containing ABCA4-1D4 tag that had been cut with restriction enzymes Afe1 and Fse1. All DNA constructs were verified by Sanger sequencing.

Scortecci J.F., Molday L.L., Curtis S.B., Garces F.A., Panwar P., Van Petegem F, & Molday R.S. (2021). Cryo-EM structures of the ABCA4 importer reveal mechanisms underlying substrate binding and Stargardt disease. Nature Communications, 12, 5902.

+ Open protocol

+ Expand

Generating HKU1 Spike Protein Constructs

Check if the same lab product or an alternative is used in the 5 most similar protocols

The full-length, codon-optimized genotype A HKU1 spike gene (Accession #: AY597011) preceded by a Kozak sequence was synthesized by GenScript (Piscataway, NJ) and cloned into pcDNA3.1(+) (Invitrogen) between HindIII and XbaI sites for eukaryotic expression. The pcDNA-HKU1A S plasmid was served as a template for the deletion constructs. A series of deletion constructs encoding HKU1 spike protein residues 14-294 (NTD), 14-687, 14-755 (S1), 295-755, 310- 677 (1A-S310-677aa), 307-687, 353-687 and 384-687 were amplified by PCR using primers listed in Supplementary Table 1 and inserted into pFastBac-1 with a hemo signal peptide at the N-terminus and a His tag at the C-terminus. Codon-optimized genotype B HKU1 S1 gene (Access#: AGT17758.1) preceded by a Kozak sequence (Supplementary Table 2) was also synthesized by GenScript (Piscataway, NJ) and cloned into pcDNA3.1 (pcDNAHKU1B S1). A DNA fragment encoding HKU1B S310-676aa (residues 310–676) was amplified by PCR using pcDNAHKU1B S1 as the template and cloned into pFastBac-1 for insect cell expression. All mutagenesis was carried out using Q5 mutagenesis kit (NEB, MA, USA). After the entire coding sequences were verified by sequencing, the sequences were cloned back into the pFastBac-1.

Ou X., Guan H., Qin B., Mu Z., Wojdyla J.A., Wang M., Dominguez S.R., Qian Z, & Cui S. (2017). Crystal structure of the receptor binding domain of the spike glycoprotein of human betacoronavirus HKU1. Nature Communications, 8, 15216.

+ Open protocol

+ Expand

Characterization of DHBV Epsilon Sequence Variants

Check if the same lab product or an alternative is used in the 5 most similar protocols

The parental DHBV16 expression vector pCD16 carries a 1.1 × DHBV16 genome (GenBank accession no. K01834; DHBV16 positions 2520–3021/1–2816) under control of the cytomegalovirus (CMV) immediate-early (IE) enhancer promoter. In its derivative pCD16_Δ3′ε the 3′ copy of Dε was made non-functional by a 40 nt deletion (DHBV16 positions 2568–2607). For in vitro transcription, pUC19T7 vectors were used which carry the Dε sequence, or derivatives thereof, under control of the bacteriophage T7 RNA polymerase promoter^{22 (link), 30 (link)}. Vectors encoding the modified Dε sequences S1 and S12 have previously been described^{22 (link), 30 (link)}. Additional mutations were introduced via conventional cloning of PCR products obtained using mutagenic primers, or via the Q5 mutagenesis kit (NEB). Generation of the SELEX vector pools is detailed in Supplementary Methods. All construct were verified by Sanger sequencing.

Gajer M., Dörnbrack K., Rösler C., Schmid B., Beck J, & Nassal M. (2017). Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming. Scientific Reports, 7, 7120.

+ Open protocol

+ Expand

Generation of Engineered Antigen-Presenting Cells

Cited 3 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

The lentiviral vector, pLv, and its associated packaging plasmids, bearing Gag/Pol, Rev and VSV-G, respectively, were purchased from Vectorbuilder (Chicago, IL, USA). Chimeric antigen receptors with or without CD28 costimulatory sequences were synthesized and cloned into a lentiviral vector by Vectorbuilder. Human CD8 A and CD8 B genes were cloned from a human cDNA library in-house. The scFv construct for TCR-like Abs [17 (link)] was produced in P.A.M.’s lab (National University of Singapore, Singapore), and TCR construct specific for HLA-A*02:01 with peptide LMP2 A_426–434 (from EBV) was a generous gift from Hans Stauss (University College London, London, United Kingdom). Single-chain trimer HLA-A*02:01-GAG was a gift from Keith Gould (Imperial College London, London, United Kingdom). Peptide mutagenesis: GAG (SLYNTVATL) to LMP2 A_426–434 (“L2”: CLGGLLTMV) was done by using a Q5 mutagenesis Kit (New England Biolabs, Ipswich, MA, USA). All the molecular cloning work was done by using an In-Fusion HD cloning kit (Clontech, Mountain View, CA, USA), and single-chain trimer MHC constructs were cloned into pLv to generate artificial antigen-presenting CHO cells [21 (link),22 (link),23 (link)].

Wu L., Brzostek J., Sankaran S., Wei Q., Yap J., Tan T.Y., Lai J., MacAry P.A, & Gascoigne N.R. (2021). Targeting CAR to the Peptide-MHC Complex Reveals Distinct Signaling Compared to That of TCR in a Jurkat T Cell Model. Cancers, 13(4), 867.

+ Open protocol

+ Expand

Site-Directed Mutagenesis of RRM1 Using Q5 Kit

Check if the same lab product or an alternative is used in the 5 most similar protocols

The PCR (polymerase chain
reaction) site-directed mutagenesis procedure was conducted using
the New England Biolabs Q₅ Mutagenesis kit. The NEB base
changer web-tool was used to design the primers, to incorporate an
amber codon (TAG) at position A35 of RRM1. PCR was performed in a
25 μL reaction mixture, following the procedure described in
the kit manual. Each reaction mixture contained 25 ng of wild-type
RRM1 template, encoded in a pET28a vector, 125 ng of forward and reverse
primers, 10 nmol of dNTPs, 2.5 units of DNA polymerase in 35 mM Tris-HCl
(pH 8.0) containing 12 mM potassium acetate, 5 mM DTT, and 0.05% Triton
X-100 in 0.05 mM EDTA. The PCR products were ligated with the Q₅ ligase master mix and transformed into DH5α E. coli cells. Purified plasmids were verified by sequencing.

Maini R., Chowdhury S.R., Dedkova L.M., Roy B., Daskalova S.M., Paul R., Chen S, & Hecht S.M. (2015). Protein Synthesis with Ribosomes Selected for the Incorporation of β-Amino Acids. Biochemistry, 54(23), 3694-3706.

+ Open protocol

+ Expand

Cloning and Fusion of SmTRPML-GCaMP6f

Check if the same lab product or an alternative is used in the 5 most similar protocols

Codon optimization, gene synthesis, and cloning (into pcDNA3.1Zeo⁽⁺⁾) of the predicted SmTRPML sequences were done by Genscript, yielding the plasmid pcSmTRPML. This codon-optimized pcSmTRPML was fused with GCaMP6f in pcDNA3.1Zeo⁽⁺⁾ at the predicted C-terminus using a strategy similar to that used elsewhere [26 ] for fusing GCaMP6f to the C-terminus of the Orai1 protein. Briefly, the pGP-CMV-GCaMP6f plasmid (Addgene) was used as a template for PCR of the full-length GCaMP6f sequence, using primers incorporating restriction sites (EcoR1/Not1) compatible with sites at the 3’ end of SmTRPML coding region of pcSmTRPML. Both pcSmTRPML and the amplified GCaMP6f coding sequence were cut with EcoR1 and Not1 and ligated to form the pcSmTRPML-GCaMP6f fusion plasmid. Due to a stop codon created in the SmTRPML-GCaMP6f linker by this process, we designed a primer for mutagenesis with the Q5 Mutagenesis Kit (NEB) that removed the stop codon and created an in-frame linker. The linker between SmTRPML and GCaMP6f contained the amino acid sequence KANSGLPCFATMVDSS. To create the SmTRPML2-GCaMP6f splice variant fusion plasmid, a primer inserting the 12 amino acid stretch found in SmTRPML2 but not in SmTRPML1 was designed and used for mutagenesis with the Q5 Mutagenesis Kit. All sequences were verified by Sanger sequencing (Eurofins).

Bais S., Norwillo A., Ruthel G., Herbert D.R., Freedman B.D, & Greenberg R.M. (2022). Schistosome TRPML channels play a role in neuromuscular activity and tegumental integrity. Biochimie, 194, 108-117.

+ Open protocol

+ Expand

STRA8 Plasmid Construction and Mutation

Check if the same lab product or an alternative is used in the 5 most similar protocols

Human STRA8 expression plasmid was obtained from Origene (RC213536). To tag STRA8 with GFP, STRA8 cDNA was inserted into the pEGFP-N1 vector (Clontech). Mutant STRA8 plasmids were generated using Q5 mutagenesis kit (New England Biolabs). All plasmids were sequenced to confirm fidelity prior to use.

Ferder I.C., Fung L., Ohguchi Y., Zhang X., Lassen K.G., Capen D., Brown D., Xavier R.J, & Wang N. (2019). Meiotic gatekeeper STRA8 suppresses autophagy by repressing Nr1d1 expression during spermatogenesis in mice. PLoS Genetics, 15(5), e1008084.

+ 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?

Revolutionizing how scientists
search and build protocols!