Gene cloning and transformation A 1.2% agarose gel was used to electrophorese the PCR product, and the band believed to include the desired gene was removed. The GeneJET Gel Extraction Kit from Thermo Scientific (Waltham, Massachusetts, United States) was used for processing. A 1500 bp fragment...
Coupling the gene synthesis procedure to in vivo homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in E. coli and used for protein crystallization. Crystals of both ...
Given recent advances in RNA synthesis and chemical stabilization, and to yet further expand the utility of CRISPRa, we set out to develop an optimized GNE-3-based synthetic gRNA platform. To evaluate the impact of chemical modifications on the efficiency of CRISPRa induced by transient delivery...
Before subjecting these strains to GEMbLeR, we assessed whether multiplexing imposed negative side effects that could compromise efficiency of the tool. First, we assessed whether implementation of multiple GEMs caused genomic instability. To this end, we grew the ySTART strain for over 100 generation...
(Fig.2a), as eukaryotic TATA-promoters are typically associated with enhanced transcription and translation efficiency75. To minimize the length of the UPEs, and thereby reduce the GEM DNA synthesis cost, we first tested the effect of reducing the promoter region to 500 bp for threeS. ...
Cloning and functional characterization of the promoter of PsSEOF1 gene from Pisum sativum under different stress conditions using Agrobacterium-mediated transient assay. Plant Signal Behav. 2014;9(9):e29626. Article PubMed PubMed Central Google Scholar Liu Q, Kasuga M, Sakuma Y, Abe H, Miura...
Kenzelmann M, Mühlemann K: Substantially enhanced cloning efficiency of SAGE (Serial Analysis of Gene Expression) by adding a heating step to the original protocol. Nucleic Acids Research. 1999, 27: 917-918. 10.1093/nar/27.3.917. Article PubMed Central CAS PubMed Google Scholar Gowda M, ...
In order to avoid experimental errors arising from variation in the quantity and integrity of the RNA template, as well as the efficiency of the cDNA synthesis and PCR amplification, a normalization step is an essential pre-requisite. Among the several proposed methods [23, 24], internal ...
assemble the final array. This strategy is both inexpensive and adaptable but requires multiple steps for cloning.cA more versatile, but expensive, strategy uses direct synthesis and ligation to build arrays. Briefly, multiple gRNA units are synthesized in tandem, and the ends of each synthesized ...
Molecular cloning and functional analysis of 4-Coumarate: CoA ligase 4 (4CL-like 1) from Fraxinus mandshurica and its role in abiotic stress tolerance and cell wall synthesis. BMC Plant Biol. 2019;19:231. https://doi.org/10.1186/s12870-019-1812-0. Article CAS PubMed PubMed Central ...