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One Step Cloning of Defined DNA Fragments from Large Genomic Clones
Scholz, Christian,Doderlein, Gabriele,Simon, Horst H. Korean Society for Biochemistry and Molecular Biol 2006 Journal of biochemistry and molecular biology Vol.39 No.4
Recently, the nucleotide sequences of entire genomes became available. This information combined with older sequencing data discloses the exact chromosomal location of millions of nucleotide markers stored in the databases at NCBI, EMBO or DDBJ. Despite having resolved the intron/exon structures of all described genes within these genomes with a stroke of a pen, the sequencing data opens up other interesting possibilities. For example, the genomic mapping of the end sequences of the human, murine and rat BAC libraries generated at The Institute for Genomic Research (TIGR), reveals now the entire encompassed sequence of the inserts for more than a million of these clones. Since these clones are individually stored, they are now an invaluable source for experiments which depend on genomic DNA. Isolation of smaller fragments from such clones with standard methods is a time consuming process. We describe here a reliable one-step cloning technique to obtain a DNA fragment with a defined size and sequence from larger genomic clones in less than 48 hours using a standard vector with a multiple cloning site, and common restriction enzymes and equipment. The only prerequisites are the sequences of ends of the insert and of the underlying genome.
Lee, Sungjun,Borys, Michael,Abbott, Patrick,Becerra, Luis Omar,Eltawil, Alaaeldin A,Jian, Wang,Malengo, Andrea,Medina, Nieves,Snegov, Victor,Wü,thrich, Christian,Scholz, Frank IOP 2017 Metrologia Vol.54 No.-
<P></P> <P>In order to show equivalence in mass standards calibration among National Metrology Institutes (NMIs) of member countries of the 'Comité international des poids et mesures' (CIPM), key comparisons (KC) of mass standards have been carried out under the auspices of the '<I>Comité Consultatif pour la Masse et les Grandeurs Apparentées</I>' (CCM). This key comparison of 5 kg, 100 g, 10 g, 5 g and 500 mg stainless steel mass standards was based on the decision of the CCM during the 12<SUP>th</SUP> meeting held in 2010 at the Bureau International des Poids et Mesures (BIPM). KRISS (Republic of Korea) and PTB (Germany) acted as pilot laboratory and co-pilot laboratory, respectively. The results were evaluated with the Monte Carlo method using measurement values based on participants' reference standards calculated following the recent BIPM amendments in 2015. Regarding participant results, VNIIM (100 g and 5 g) were not consistent with the key comparison reference values within their expanded uncertainties with the coverage factor, <I>k</I> = 2.</P> <P></P> <H2>Main text</H2> <P> To reach the main text of this paper, click on <A HREF='http://www.bipm.org/utils/common/pdf/final_reports/M/M-K7/CCM.M-K7.pdf'>Final Report</A>. Note that this text is that which appears in Appendix B of the BIPM key comparison database <A HREF='http://kcdb.bipm.org/'>kcdb.bipm.org/</A>.</P> <P>The final report has been peer-reviewed and approved for publication by the CCM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).</P>