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发布于:2018-7-19 23:10:18  访问:3 次 回复:0 篇
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Hose sequences are based on the sequence of gene lti2 (40). Restructuring
Restriction fragments subcloned into pBluescript SK( ) (Stratagene, La Jolla, Calif.) had been subjected to automated sequencing (Applied Biosystems Inc., Foster City, Calif.) on each strands in the DNA.Database comparisons and alignments in the translated sequences have been performed by utilizing the default settings in the K-Ras(G12C) inhibitor 12 biological activity algorithm created by Altschul et al. pAHS5, pAHS6, and pAHS7 have been introduced into SA6 by conjugation, with selection on AAN containing 10 g of spectinomycin ml 1. Nucleotide sequence accession quantity. The sequence of orrA reported in this paper has been submitted to GenBank beneath accession no.Hose sequences are according to the sequence of gene lti2 (40). Restructuring the AB5 mutant and second-site mutagenesis. So that you can perform second-site mutagenesis by insertion of a transposon, plasmid pRL386a (Fig. 1b) was transferred to AB5 by conjugation. Quite a few single recombinants had been chosen on erythromycin, grown in liquid culture, and plated on medium containing 5 sucrose to pick for double recombinants lacking the sacB gene. Survivors that have been presumptively identified as double recombinants by their sucrose-resistant, erythromycin-resistant, neomycin-sensitive, and spectinomycin-sensitive phenotype have been confirmed as double recombinants by Southern analysis and showed an induction of luciferase by salt that was equivalent for the induction shown by AB5 (data not shown). One particular double recombinant, designated AB5DR386a, was subjected to second-site mutagenesis by use of the transposonbearing plasmid pRL1058 (45). Following mutagenesis, Nuclepore membranes bearing colonies derived from transposition were transferred from AAN medium to AAN medium supplemented with 0.1 M NaCl. Colonies with out elevated luciferase activity have been identified by overlaying images of luminescence 5 h following transfer to salt-containing medium, with pictures portraying the positions of colonies. One particular derivative hence identified, which was designated SA6, was selected for further experimentation. Isolation of a genomic clone of SA6. An EMBL3 lambda library (six) was screened with pRL871, which had been labeled in a random primer labeling reaction (Promega, Madison, Wis.). Phage DNA from good plaques was isolated from plate lysates (39); digested with SalI, internet sites for which bracket the cloned insert; and ligated PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22792370 into the SalI site of pRL171 (15). One particular resulting plasmid was designated pAHS1. Restriction fragments subcloned into pBluescript SK( ) (Stratagene, La Jolla, Calif.) have been subjected to automated sequencing (Applied Biosystems Inc., Foster City, Calif.) on each strands of your DNA.Database comparisons and alignments with the translated sequences have been performed by utilizing the default settings from the algorithm created by Altschul et al. (two) at the National Center for Biotechnology Information and facts together with the BLAST network service. The molecular weight of OrrA was calculated by the ExPASy webserver in the Swiss Institute of Bioinformatics, Geneva, Switzerland. Reconstruction of mutant SA6. Plasmid pRL1380 was transferred to AB5DR386a, and recombinants were chosen on medium containing 400 g of neomycin ml 1. Numerous recombinants had been grown in liquid culture and were then plated on agar medium containing five (wt/vol) sucrose. Protein analysis. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/23030295 Cultures of wild-type PCC 7120, AB5DR386a, and SA6 were induced for eight h by exposure to AAN medium supplemented with 75 mM NaCl and 75 mM KCl.
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