nru i  (New England Biolabs)

Journal: Molecular and Cellular Biology

Article Title: Mice Devoid of Fer Protein-Tyrosine Kinase Activity Are Viable and Fertile but Display Reduced Cortactin Phosphorylation

doi: 10.1128/MCB.21.2.603-613.2001

Figure Lengend Snippet: Domain structure of Fer and targeting strategy of fer locus. (A) Fer consists of three CC motifs, a central SH2 domain, and a kinase domain. The catalytic subdomains are shown, with subdomain IX encoded by exon 19 contained within genomic clone 3B. The targeting vector contains a 6-kbp long arm of homology, followed by the PGK-neo cassette, a 0.9-kbp short arm of homology, and a PGK-tk cassette. The D743R mutation was generated within exon 19, which also introduces an Nru I (N) site. A schematic of the targeted allele following homologous recombination is shown, with positions of PCR primers and Southern blot probe indicated. Positions of Xba I (X), Xho I (Xh), Nhe I (Nh), Eco RI (E), and Sac I (S) sites are shown. Sites in parentheses were destroyed. (B) Southern blot analysis of Sac I-digested genomic DNA from animals that were wild type (+/+), heterozygous (+/−), or homozygous (−/−) for fer D743R . Hybridization with a probe located 3′ to the sequence used for the short arm of homology reveals bands of 4.0 kbp for the wild-type allele and 5.8 kbp for the fer D743R allele. (C) Genotyping PCR analysis of genomic DNA from +/+, +/−, or −/− animals. The resulting 871-bp fragment is resistant to Nru I digestion in wild-type samples, yields additional 605- and 266-bp bands in heterozygous samples, and yields only 605- and 266-bp fragments in homozygous samples.

Article Snippet: The PCR-generated 871-bp fragment was subsequently digested with Nru I (NEB), yielding bands of 605 and 266 bp in the presence of the D743R mutation.

Techniques: Plasmid Preparation, Mutagenesis, Generated, Homologous Recombination, Polymerase Chain Reaction, Southern Blot, Hybridization, Sequencing

Journal: Viruses

Article Title: A Novel Plasmid DNA-Based Foot and Mouth Disease Virus Minigenome for Intracytoplasmic mRNA Production

doi: 10.3390/v13061047

Figure Lengend Snippet: Construction of recombinant plasmids. ( A ) The DNA cassette containing T7 promoter and FMDV O189 5′UTR in pKLS1 vector was removed by digesting with the restriction enzymes, Nru I and Stu I, and placed upstream of FMDV O189 3′UTR, in plasmid pUC57_3′UTR, resulting in the pKLS3 vector. The complete pKLS3 construct is an FMDV minigenome containing the essential FMDV cis-acting elements for viral replication/transcription and translation and a Stu I site for foreign gene insertion. ( B ) The enhanced green fluorescence protein (GFP) gene was inserted into pKLS3 at the Stu I site between FMDV 5′and 3′UTRs to generate a minigenome with a transfection reporter pKLS3_GFP.

Article Snippet: The DNA cassette containing the sequence T7 promoter-pol I promoter-FMDV O189 5′ UTR in 5′ to 3′ direction was excised with Nru I/Stu I restriction enzymes (New England Biolab, Ipswich, MA, USA) from the pKLS1 vector and then ligated into pUC57_3′UTR upstream the 3′UTR, to create pKLS3 vector ( A).

Techniques: Recombinant, Plasmid Preparation, Construct, Fluorescence, Transfection

Journal: Infection and Immunity

Article Title: Isolation of Chlamydia pneumoniae Clonal Variants by a Focus-Forming Assay

doi: 10.1128/IAI.70.10.5827-5834.2002

Figure Lengend Snippet: (A) Genomic map of the tyrP region. Sequencing data revealed a 1,649-bp polymorphism containing a duplication of the tyrP and ycc ). Lines indicate the cutting sites of Nru I resulting in 3,974- and 5,623-bp fragments, respectively. The bar indicates the region amplified by the duplication-PCR. (B) Southern blot analysis after Nru I digestion. The MUL-250 wild-type strain shows two bands of 3,974 and 5,623 bp, respectively, indicating the presence of both a single tyrP copy population and a double tyrP copy population. Clones obtained by the focus assay showed either a double copy (clone 38) or a single copy (clone 41). HeLa DNA was used as a negative control (line 4). (C) Agarose gel analysis of duplication-PCR products (93 bp). Screening of 60 C. pneumoniae strain MUL-250 clones revealed a single clone (clone 38) containing a duplicated tyrP gene as indicated by a positive PCR product. A negative duplication-PCR for clone 41 indicates the presence of a single tyrP copy only. The MUL-250 wild-type strain was used as a positive control, and H 2 O was used as a negative control (lane 4).

Article Snippet: Two hundred nanograms of DNA prepared from each clone and uninfected HeLa cells were digested with Nru I (New England BioLabs, Beverly, Mass.), separated on a 1% agarose gel, and transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, Great Britain) by capillary blotting using the neutral transfer protocol as described by the manufacturer.

Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Southern Blot, Clone Assay, Negative Control, Agarose Gel Electrophoresis, Positive Control

Journal: Cellular and Molecular Life Sciences

Article Title: Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense

doi: 10.1007/s00018-016-2173-6

Figure Lengend Snippet: Loss or mutation of AT1 in the resistant lines . a Genomic 454 reads of STIB900-P and STIB900-M mapped to STIB900 visualized for the AT1 locus on chromosome 5 using BamView [ 59 ] (smaller scale for STIB900-M). There is a deletion of AT1 in STIB900-M and a coding point mutation in STIB900-P ( red ). b AT1 PCR products (1636 bp) were amplified from genomic DNA of STIB900 and STIB900-P, and digested with the endonuclease Nru I. G1288C mutant alleles are cut to fragments of 1339 and 257 bp. STIB900-P appears to be heterozygous for the mutation

Article Snippet: The PCR product was purified on silica-membrane columns (Nucleospin gel and PCR clean up, Macherey–Nagel) and digested with Nru I (New England Biolabs), run on a 1.5 % agarose gel and visualized with ethidium bromide.

Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification