The location of sequencing primer annealing sites is indicated (S

The location of sequencing primer annealing sites is indicated (SS1 and SS2). The I-SceI recognition sites are shown flanking the cloning region. (B) DNA sequences of the pDOC-K, pDOC-H, pDOC-F, pDOC-P and pDOC-G inserts. Sequences specific to each plasmid are shown in the open box. The first codon of the epitope tags are highlighted in grey, and the stop codons are indicated. The following primer annealing sites are indicated: SS1 and SS2, used to sequence plasmid derivatives pre-recombination;

K-FWD, used for amplifying PCR products from Verubecestat clinical trial pDOC-K for generating gene deletions; CC1 and CC2, used for generating PCR products in order to confirm recombination; P-REV, used to generate PCR products for cloning into pDOC-C pre-recombination. The Flp recognition sequences are shown (Flp1 and Flp2), flanking the kanamycin cassette. The cloning regions, CR1 and CR2 are shown, adjacent to the I-SceI recognition sites. G-DOC recombineering protocol For generating gene:epitope tag fusions, the epitope tag and kanamycin cassette are amplified by PCR, using the relevant pDOC plasmid as a template.

A schematic outline of the cloning strategy for generating gene:epitope tag fusions is shown in Figure 3, panel A. The clockwise primer used for the PCR {Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|buy Anti-infection Compound Library|Anti-infection Compound Library ic50|Anti-infection Compound Library price|Anti-infection Compound Library cost|Anti-infection Compound Library solubility dmso|Anti-infection Compound Library purchase|Anti-infection Compound Library manufacturer|Anti-infection Compound Library research buy|Anti-infection Compound Library order|Anti-infection Compound Library mouse|Anti-infection Compound Library chemical structure|Anti-infection Compound Library mw|Anti-infection Compound Library molecular weight|Anti-infection Compound Library datasheet|Anti-infection Compound Library supplier|Anti-infection Compound Library in vitro|Anti-infection Compound Library cell line|Anti-infection Compound Library concentration|Anti-infection Compound Library nmr|Anti-infection Compound Library in vivo|Anti-infection Compound Library clinical trial|Anti-infection Compound Library cell assay|Anti-infection Compound Library screening|Anti-infection Compound Library high throughput|buy Antiinfection Compound Library|Antiinfection Compound Library ic50|Antiinfection Compound Library price|Antiinfection Compound Library cost|Antiinfection Compound Library solubility dmso|Antiinfection Compound Library purchase|Antiinfection Compound Library manufacturer|Antiinfection Compound Library research buy|Antiinfection Compound Library order|Antiinfection Compound Library chemical structure|Antiinfection Compound Library datasheet|Antiinfection Compound Library supplier|Antiinfection Compound Library in vitro|Antiinfection Compound Library cell line|Antiinfection Compound Library concentration|Antiinfection Compound Library clinical trial|Antiinfection Compound Library cell assay|Antiinfection Compound Library screening|Antiinfection Compound Library high throughput|Anti-infection Compound high throughput screening| amplification is designed so that it contains between 25-50 bp of homology to the 3′ end of the target gene (H1), not including the Ferroptosis activation stop codon, followed by 20 bp of sequence which anneals to the epitope tag sequence on the pDOC plasmid. This should be designed so that, after recombination with the target gene on the chromosome, the gene will be in frame with the coding sequence of the epitope tag. The downstream primer is designed so that it contains 25-50 bps of homology to the DNA sequence immediately downstream of the target gene (H2) and the primer sequence P-REV. The two primers are also designed with a restriction site at the 5′ end, so that, Oxymatrine after amplification by PCR, the DNA product can be cloned into the cloning region of pDOC-C, between the two I-SceI

sites. Figure 3 Schematic of pDOC based recombination. PCR products are generated for gene coupling (A) or for gene deleting (B) and cloned into pDOC-C. Homologous regions (H1-4) on the PCR product recombine with the target gene on the chromosome. Recombinant clones are then checked by PCR using primers annealing to the CC1 and CC2 sequences, and sequences adjacent to the homology regions. For generating gene deletions, the kanamycin cassette from pDOC-K, is amplified by PCR. A schematic outline of the cloning strategy for generating gene deletions is shown in Figure 3, panel B. The clockwise primer used for the PCR amplification is designed so that it contains between 25-50 bp of homology to the DNA immediately upstream of the start of the gene (H3), followed by 20 bp of sequence which anneals to the K-FWD sequence on pDOC-K.

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