Lr recombination reaction

Manufactured by Thermo Fisher Scientific

Sourced in United States

The LR recombination reaction is a method used to transfer DNA fragments between compatible vectors. It facilitates the directional cloning of DNA sequences into an expression vector. The reaction relies on the site-specific recombination properties of bacteriophage lambda to efficiently join DNA fragments.

Automatically generated - may contain errors

Lab products found in correlation

38 protocols using lr recombination reaction

Markerless Gene Deletion Protocol

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

Approximately 700 bp flanking the gene of interest were amplified and fused with assembly PCR using 5′ linkers added to the internal primers. The assembled product was cloned into a Gateway entry vector and transferred into pAK31GW (Gateway-compatible sacB counterselectable suicide vector) using the LR recombination reaction (Life Technologies). The resulting plasmid was used to generate markerless in-frame deletions.

Clark I.C., Melnyk R.A., Youngblut M.D., Carlson H.K., Iavarone A.T, & Coates J.D. (2015). Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1. mBio, 6(3), e00282-15.

+ Open protocol

+ Expand

Multifragment Fusion Gene Construction

Cited 1 time

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

The fusion gene constructs were generated using the Gateway multifragment recombination technique, as previously described (12 (link)). Briefly, fusion gene construction was based on PCR using sequence-verified open reading frames (ORFs) corresponding to each fusion gene fragment obtained from the ORFeome collaboration (www.orfeomecollaboration.org), Mammalian Gene Collection, and commercial ORF sources (Ultimate ORF Clones; Life Technologies) as PCR template. PCR primers were generated to amplify the desired fragments (left and right gene arms of each fusion) with terminal Gateway (Life Technologies) recombination sequences. The left and right arm PCR products were incorporated into pDONR vectors (Life Technologies) and pFUSE-B vector (Addgene, plasmid no. 97185), respectively, through BP recombination (Life Technologies), followed by multifragment recombination into the pFUSE-DEST_R1R4 vector (Addgene, plasmid no. 97186) through LR recombination reaction (Life Technologies) following the manufacturer’s recommendations. The reaction mixtures were incubated at room temperature overnight and subsequently transformed into STBL3 (Life Technologies) competent bacteria.

Li J., Lu H., Ng P.K., Pantazi A., Ip C.K., Jeong K.J., Amador B., Tran R., Tsang Y.H., Yang L., Song X., Dogruluk T., Ren X., Hadjipanayis A., Bristow C.A., Lee S., Kucherlapati M., Parfenov M., Tang J., Seth S., Mahadeshwar H.S., Mojumdar K., Zeng D., Zhang J., Protopopov A., Seidman J.G., Creighton C.J., Lu Y., Sahni N., Shaw K.R., Meric-Bernstam F., Futreal A., Chin L., Scott K.L., Kucherlapati R., Mills G.B, & Liang H. (2022). A functional genomic approach to actionable gene fusions for precision oncology. Science Advances, 8(6), eabm2382.

+ Open protocol

+ Expand

Generating Elk1 Lentiviral Vectors for Transduction

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

A lentiviral vector encoding Elk1 was generated by Gateway Technology (Thermo Fisher Scientific) as described previously (Suzuki et al., 2010). Briefly, Elk1 cDNA (a gift from H. Sugimoto, Dokkyo Medical University) was subcloned into the pENTR vector (Thermo Fisher Scientific) and subsequently transferred into the pCSII‐EF‐RfA lentiviral expression vector (a gift from H. Miyoshi, Keio University) by the LR recombination reaction (Thermo Fisher Scientific). GFP‐bearing CS‐CDF‐CG‐PRE plasmid was used as a control lentiviral vector. The 293FT cells were co‐transfected with the expression plasmids and packaging plasmids (pCMV‐VSV‐G‐RSV‐Rev and pCAG‐HIVgp) using Lipofectamine 2000 (11668019; Thermo Fisher Scientific). The viral supernatants were collected 48 h after transfection. For viral infection, 5.0 × 10⁴ TEC cells per well in 12‐well tissue culture plates were infected with lentiviral particles.

Akatsu Y., Takahashi N., Yoshimatsu Y., Kimuro S., Muramatsu T., Katsura A., Maishi N., Suzuki H.I., Inazawa J., Hida K., Miyazono K, & Watabe T. (2019). Fibroblast growth factor signals regulate transforming growth factor‐β‐induced endothelial‐to‐myofibroblast transition of tumor endothelial cells via Elk1. Molecular Oncology, 13(8), 1706-1724.

+ Open protocol

+ Expand

Gateway Cloning for Protein Expression

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

The coding sequence of the genes of interest were amplified using PCR and cloned to pDONR221 via a Gateway BP reaction (Thermo Fisher Scientific, Waltham, MA, USA). Primer sequences used in this study are listed in Table S1. For generating N- and C-terminal GFP-tagged proteins driven by CaMV 35S promoter, genes were cloned to pK7WGF2 and pK7GWF2, respectively [61 (link)], through an LR recombination reaction (Thermo Fisher Scientific).
To generate gene-overexpressing constructs, cloning vectors that obtained the genes of interest were recombined with a pKm43GW-RedRoot destination vector which contains DsRed as a marker gene [62 (link)]. The CaMV 35S promoter was used to drive DsRed and the genes of interest.
To generate constructs for the split-ubiquitin membrane yeast two-hybrid assay, the coding sequence of MtNLA1, MtNLA3 variants and SPX domain of MtNLA3.1 (1–210 a.a.) were cloned to the BamHI and EcoRI sites of the prey vectors, pDL-Nx and pDL-xN. The coding sequence of MtPT1 and MtPT4 were cloned to the XbaI and StuI sites, respectively, of the bait vector, pAMBV4 (Dualsystems Biotech, Schlieren, Switzerland).

Lin W.Y., Yang H.N., Hsieh C.Y, & Deng C. (2023). Differential Responses of Medicago truncatula NLA Homologs to Nutrient Deficiency and Arbuscular Mycorrhizal Symbiosis. Plants, 12(24), 4129.

+ Open protocol

+ Expand

Human Gene Coding Sequence Library Construction

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

Protein coding sequences (CDS) for all human genes were downloaded from RefSeq database and the longest isoform of each gene used for oligonucleotide design. The protein sequences were first encoded as DNA bases using random codons, which were then divided into 270 bp regions tiling across the entire CDS with 135 bp overlaps between neighboring oligonucleotides and with flanking 15 bp primers as described (Xu et al., 2016 (link)). The DNA was amplified by PCR using specific primers that included attB sites to allow the products to be cloned into pDONR223 via a Gateway BP recombination reaction and subsequently into the the lentiviral GPS vector pHAGE-GPS3.0-DEST via an LR recombination reaction (Thermo Fisher Scientific). At least 100-fold representation of the library was maintained at each cloning step.

Koren I., Timms R.T., Kula T., Xu Q., Li M.Z, & Elledge S.J. (2018). The Eukaryotic Proteome is Shaped by E3 Ubiquitin Ligases Targeting C-terminal Degrons. Cell, 173(7), 1622-1635.e14.

+ Open protocol

+ Expand

Cloning parB Genes into pET-His-MBP-TEV

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

The parB genes were recombined into a Gateway-compatible destination vector pET-His-MBP-TEV-DEST (Jalal et al., 2019 (link)) via an LR recombination reaction (ThermoFisher). For LR recombination reactions: 1 µL of purified pENTR::parB (~100 ng/µL) was incubated with 1 µL of the destination vector pET-His-MBP-TEV-DEST (~100 ng/µL), 1 µL of LR Clonase II enzyme mix, and 2 µL of water in a total volume of 5 µL.

Jalal A.S., Tran N.T, & Le T.B. (2020). ParB spreading on DNA requires cytidine triphosphate in vitro. eLife, 9, e53515.

+ Open protocol

+ Expand

Cloning and Mutagenesis of UBASH3A and CBLB

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

A cDNA of the full-length human UBASH3A was cloned into the Gateway pDONR221 vector (Thermo Fisher Scientific), and the resultant plasmid was used as the template for QuikChange site-directed mutagenesis (Agilent Technologies). The K202R mutation was generated using the primers 5’-gaccttggcccacaggttctacccccacc-3’ and 5’-ggtgggggtagaacctgtgggccaaggtc-3’. The W279A mutation was generated using the primers 5’-gaagccagcgagggcgcggtgattgggatctc-3’ and 5’-gagatcccaatcaccgcgccctcgctggcttc-3’. Next, we performed the LR recombination reaction (Thermo Fisher Scientific) to move the cDNA sequences of wild-type, K202R and W279A UBASH3A from the pDONR221 vector to the Gateway pcDNA3.1/nV5-DEST expression vector. The accuracy of all UBASH3A inserts was confirmed by Sanger sequencing.
A pBluescriptR plasmid containing the cDNA sequence of full-length human CBLB was purchased from Thermo Fisher Scientific, and the CBLB cDNA sequence was cloned into the pCMV-Tag2 expression vector (Agilent Technologies) using the restriction enzymes PstI and XhoI (New England BioLabs).
HEK293T cells were transfected with the X-tremeGENE HP DNA Transfection Reagent (Roche), following the manufacturer’s protocol.

Ge Y., Paisie T.K., Chen S, & Concannon P. (2019). UBASH3A regulates the synthesis and dynamics of T-cell receptor-CD3 complexes. Journal of immunology (Baltimore, Md. : 1950), 203(11), 2827-2836.

+ Open protocol

+ Expand

Modulating Protein Interactions in Cells

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

pcDNA3-HA-Rbx1 (#19897), pcDNA3-Myc-Cul3 (#19893), Cul3-H2M/H5M (#21591), Cul3-deltaN41 (#21590), pcDNA3-DN-hCUL3-Flag (#15820), Flag-β-TrCP (#10865), HA-tagged Ubiquitin (#17608) were obtained from Addgene. pcDNA3-HA-APOBEC3G was a kind gift from Dr. Reuben Harris (University of Minnesota). pCMV6-Entry encoding HIV-1-Vif (VC101719), HBV X protein (VC102194), HPV E7 protein (VC101903) were purchased from Origene. Point mutations and pre-mature stop codons in Wa-NSP1 were introduced into pENTR221 vector using QuikChange II Site-Directed Mutagenesis Kit (Agilent) and shuttled into pG-LAP6 destination vector using LR recombination reaction (Thermo Fisher).

Ding S., Mooney N., Li B., Kelly M.R., Feng N., Loktev A.V., Sen A., Patton J.T., Jackson P.K, & Greenberg H.B. (2016). Comparative Proteomics Reveals Strain-Specific β-TrCP Degradation via Rotavirus NSP1 Hijacking a Host Cullin-3-Rbx1 Complex. PLoS Pathogens, 12(10), e1005929.

+ Open protocol

+ Expand

Adenovirus Production for Primary Hepatocytes

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

The coding sequence of mouse JCAD was cloned and ligated with pEntry shuttle vector (Invitrogen). The pEntry‐JCAD clone was recombined with the adenoviral vector pAd/PL‐DEST by an LR recombination reaction (Invitrogen), and pEntry and pAd/PL‐DEST vectors were kind gifts from Dr. Jieliang Chen in the Key Laboratory of Medical Molecular Virology, Fudan University Shanghai Medical College. After PacI digestion, the linearised plasmid was transfected into HEK293 cells and amplified for three rounds. The adenoviruses were purified by discontinuous iodixanol gradient centrifugation. The TCID50 of purified adenoviruses was determined by endpoint dilution assay, and the primary hepatocytes were infected with an multiplicity of infection (MOI) at 20−40.

Zhang L., Yang Y., Xie L., Zhou Y., Zhong Z., Ding J., Wang Z., Wang Y., Liu X., Yu F, & Wu J. (2024). JCAD deficiency delayed liver regenerative repair through the Hippo–YAP signalling pathway. Clinical and Translational Medicine, 14(3), e1630.

+ Open protocol

+ Expand

V5-tagged Protein Expression and IP in mES Cells

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

For V5-tagged protein expression and immunoprecipitation, mouse ES cells were electroporated using the Neon transfection system (Invitrogen) with an episomally-replicating vector (pCAG-GW-V5-Hygro) encoding expression of a C-terminal V5 tagged ORF driven by a CAG promoter. ORFs were obtained from the DNASU plasmid repository as Gateway entry clones and inserted into pCAG-GW-V5-Hygro using an LR recombination reaction (Invitrogen). Transfected cells were selected on 125ug/mL Hygromycin B (Invitrogen) to generate stably expressing lines.

McHugh C.A., Chen C.K., Chow A., Surka C.F., Tran C., McDonel P., Pandya-Jones A., Blanco M., Burghard C., Moradian A., Sweredoski M.J., Shishkin A.A., Su J., Lander E.S., Hess S., Plath K, & Guttman M. (2015). The Xist lncRNA directly interacts with SHARP to silence transcription through HDAC3. Nature, 521(7551), 232-236.

+ 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!