In fusion hd pcr cloning kit

Chicken genomic DNA was extracted from the liver of a Cobb broiler chicken using Quick-gDNA Microprep Kit (Zymo Research, Irvine, CA) according to the manufacturer's recommendations. A series of AvBD9 gene promoter constructs were cloned from chicken genomic DNA using CloneAmp HiFi PCR Premix (Takara Bio USA, Mountain View, CA) with different forward primers paired with a common reverse primer (Table 1). It is noted that the 5′-end of gene-specific reverse primer begins at the third nucleotide upstream of the start codon of the AvBD9 mRNA (GenBank accession number NM_001001611). PCR products were then cloned into a KpnI-linearized luciferase reporter vector, pGL4.21[luc2P/Puro] (Promega, Madison, WI), using a ligation-independent In-Fusion HD PCR Cloning Kit (Takara Bio USA). The presence of the insert in each recombinant plasmid was confirmed with direct Sanger sequencing. Recombinant plasmids were propagated in Stellar E. coli HST08 competent cells (Takara Bio USA) and purified with QIAprep Spin Plasmid Miniprep Kit (Qiagen, Germantown, MD) for transient transfections as described below.

To investigate subcellular localization, the coding sequences of OSAT.v7_FAR1.Chr10.2 (hereafter called OFF) and OsRH36 were amplified and fused with GFP and mCherry, respectively, in the HindIII-digested pGreen vector using the In-Fusion HD PCR Cloning Kit (Takara). The plasmids were transformed into Agrobacterium tumefaciens GV3101 individually and used for tobacco infiltration assays as described below. To generate a knockout mutant of OFF using CRISPR/Cas9 genome editing, we designed a single-guide RNA through CRISPRdirect (http://crispr.dbcls.jp/) (31 (link)). Designed oligomers were synthesized, and annealed oligomer was ligated into the BsaI-digested pRGEB32 binary vector (Addgene plasmid ID: 63142). After the plasmid was transformed into A. tumefaciens LBA4404, stable transformation of rice was performed using cv. Dongjin through the Agrobacterium-mediated co-cultivation method as described in Lee et al. (32 ). The primers for vector construction are listed in Supplementary Table S9.