Sacb selection
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Sacb selection
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WebNov 19, 2014 · The cat-sacB cassette facilitated generation of multiple successive mutations in A. pleuropneumoniae using the two-step transformation protocol. Cm selection was very stringent, and all Cm-resistant clones tested were confirmed to contain the cat-sacB cassette by PCR (data not shown).
WebApr 30, 2016 · I have a problem getting my sacB counter-selection to work. sacB sequence (obtained from PYUB870) was cloned into the MCS of pGEM-T, transformed into E. coli … WebSep 19, 2024 · sacB counter-selection of the second homologous recombination Colonies growing on the BHI/Tc100 plates were checked by colony PCR using the pEX18 universal forward primer and a primer specific to the genomic DNA following the cloned 500 bp downstream sequence (5′ ACATCTGCACCAGGTCGTC 3′), as illustrated in Fig. 4.
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WebOct 1, 2024 · A knockout vector (pMTL70621-SacB) was also constructed carrying the sacB counter selection marker. Through provision of the appropriate mutant alleles, the vector was subsequently used to delete genes encoding restriction enzymes by selecting cells able to grow in the presence of 15% ( w / v) sucrose (see Supplementary Information).
WebMay 24, 2024 · We tested counter-selection efficiency in multiple Salmonella loci using the commonly used markers sacB or I-sceI (Fig. 1b). sacB codes for levan sucrase, which … probation service cardiff jobsWebI have a problem getting my sacB counter-selection to work. sacB sequence (obtained from PYUB870) was cloned into the MCS of pGEM-T, transformed into E. coli and plated on 1) 5% sucrose LB agar ... probation service cheshireWebMay 29, 2016 · Selection is via growth on agar plates containing sucrose. As sucrose is lethal to cells when the sacB gene product is expressed, the only cells that survive are those in which a second recombination event has occurred, whereby the sucrose sensitivity gene, in addition to the antibiotic resistance gene, has been recombined out of the chromosome … regalplanung softwareUnlike positive selection, negative selection means you’re selecting for the loss of a gene product - usually something toxic. This gene is found on the original plasmid and either the insertion of a DNA fragment within the gene or loss of the gene alleviates its toxic effect. Negative selection is sometimes called … See more In positive selection, only cells that contain a specific gene survive. The rest do not grow. While there are many ways to design your positive selection strategy, here are a few to consider. See more At this point in the blog post, you may have realized that positive and negative selection strategies adopt naturally occurring genes for … See more probation service center dakota countyhttp://lowepowerlab.ucdavis.edu/protocols/gene_deletion.html probation service complaints procedureWebJan 1, 2012 · B. subtilis sacB gene has been used as a selection marker for gene disruption in the chromosome of E. coli ( Gay et al., 1985) and C. glutamicum ( Schafer et al., 1994 ). In most cases, when used in C. glutamicum, the sacB gene was cloned together with its 463 bp upstream region ( Steinmetz et al., 1985 ). probation service chathamWebMar 22, 2016 · Sucrose-LB agar (5% concentration) was used for sacB gene-based counter selection in mutant strains’ construction. 4.2. Construction of Plasmids and Mutations. The primers designed in this study are listed in Table S1. The protocol of DNA manipulations was performed as previous described . probation service chesterfield