Journal: Journal of Korean Medical Science

Article Title: Clarithromycin-Based Standard Triple Therapy Can Still Be Effective for Helicobacter pylori Eradication in Some Parts of the Korea

doi: 10.3346/jkms.2014.29.9.1240

Figure Lengend Snippet: Restriction fragment length polymorphism analysis of 23S rRNA amplicons: ( A ) digestion with Bsa I and ( B ) digestion with Bbs I. The A2144G mutations are observed in lanes 1 to 2, but not in lanes 3 to 5. Note that the A2143G mutation detected by digestion with Bbs I was not detected in any of the strains studied. Lanes 3 to 5 reveal the T2183C mutation, as assessed by DNA sequencing. Lane M, 100 bp DNA size markers (indicated to the left of the gels in base pairs); lane C, H. pylori ATCC 43504; lane 1 to 5, clarithromycin-resistant H. pylori strains.

Article Snippet: Amplicons (424 bp each) of the 23S rRNA gene were digested with either Bsa I (New England BioLabs, Beverly, MA, USA) for 14 hr at 50℃ or Bbs I (New England BioLabs) for 14 hr at 37℃ to detect the A2144G and A2143G mutations, respectively ( ).

Techniques: Mutagenesis, DNA Sequencing

Journal: Molecular Pain

Article Title: Peripheral non-viral MIDGE vector-driven delivery of ?-endorphin in inflammatory pain

doi: 10.1186/1744-8069-5-72

Figure Lengend Snippet: Construction of MIDGE vectors . (A) POMC exon 2-3 cDNA, encoding the signal peptide, adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH) and β-endorphin (END), was spliced into the pMOK plasmid between the SacI and KpnI restriction sites. The pMOK plasmid contains a kanamycin resistance gene, a cytomegalovirus promoter (PCMV), a chimeric intron and simian virus (SV) 40 polyadenylation (pA) site. The POMC-MIDGE-NLS was derived from the pMOK-POMC plasmid by cutting it with Eco31I, ligation with hairpin ODNs at both ends, and coupling of nuclear localization sequence (NLS) to one of the hairpin ODN. The other vectors were prepared analogously by modifying POMC gene as follows: (B) END sequence was removed to obtain control MIDGE-NLS; (C) ACTH and MSH fragments were removed while END sequence was preserved in its original place to obtain 1xEND-MIDGE-NLS encoding 1 copy of END; (D) ACTH fragment was removed, original END sequence was preserved and MSH fragment was replaced with an additional sequence of END to obtain 2xEND-MIDGE-NLS encoding 2 copies of END; (E) original END sequence was preserved and the ACTH and MSH fragments were replaced with additional END sequences to obtain 3xEND-MIDGE-NLS encoding 3 copies of END.

Article Snippet: The product was cut with Eco31I and SacI, ligated and cloned into the pMOK-POMC1xEND plasmid after digestion with SacI and BpiI (Fermentas).

Techniques: Plasmid Preparation, Derivative Assay, Ligation, Sequencing

Journal: PLoS ONE

Article Title: Efficient Generation of Recombinant Influenza A Viruses Employing a New Approach to Overcome the Genetic Instability of HA Segments

doi: 10.1371/journal.pone.0116917

Figure Lengend Snippet: Construction strategy of the low-copy pMKP ccd B reverse genetic vector to clone unstable HA segments of IAV. The resulting vector is a hybrid of pSMART-LC-Kan and the Pol-I/ ccd B/Pol-II cassette of pMP ccd B with either AarI or Bsa I or BsmB I sites for restriction/cloning of the desired insert. Briefly, the sequence representing the Pol-I/ ccd B/Pol-II cassette excised from pMP ccd B by digestion with BsrB I were blunt end phosphorylated, and ligated into the EcoR V linearized pSMART-LC-Kan plasmid to generate pMKP ccd B. The pMKP ccd B was suitable to clone and maintain the genetically unstable RT-PCR product of H9N2/SA- and H5N1/VSVRI-HA. Transcriptional terminator (T); Repressor of Primer (ROP); low copy origin of replication (Ori); kanamycin resistance gene (Kanr), ampicillin resistance gene (Ampr).

Article Snippet: The positive and correct vector/insert constructs were digested with Bsa I-HF (NEB, Germany) for 15 min at 37°C.

Techniques: Plasmid Preparation, Clone Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction

Journal: Jundishapur Journal of Microbiology

Article Title: Detection of 23SrRNA Mutations Strongly Related to Clarithromycin Resistance in Helicobacter pylori Strains Isolated From Patients in the North of Iran

doi: 10.5812/jjm.29694

Figure Lengend Snippet: Gel Electrophoresis of Digested 429 bp Polymerase Chain Reaction Product of 23S rRNA Gene With BsaI and MboII Enzymes Lane 1, DNA ladder (100 bp); Lane 2, clarithromycin resistant strain digested with BsaI at two sites; Lane 3 and 4, wild types of H. pylori (susceptible to clarithromycin) digested with BsaI, with one digestion site; Lane 5, wild type (susceptible to clarithromycin) treated with MboII with no digestion site.

Article Snippet: To detect the A-G point mutations at positions 2142 and 2143, the PCR products were digested with the MboII and BsaI (MBI Fermentas, Lithuania) restriction enzymes, respectively, according to the manufacturer’s instructions, and analyzed on 2% agarose gel containing Sybrsafe.

Techniques: Nucleic Acid Electrophoresis, Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

doi: 10.1371/journal.pone.0005553

Figure Lengend Snippet: Assembly of a GFP construct from 10 plasmids. (A) Construct maps. Input modules contain a core region C flanked by BsaI restriction sites in opposite orientation composed of a recognition site (B, ggtctcn, B , ngagacc) and a 4 nucleotide cleavage site (boxes flanking the core region). pX-LacZ, acceptor vector. pGFPi, resulting construct. Restriction sites for AvrII and XmaI are shown as white arrows. (B) Ethidium bromide-stained gel with products obtained by restriction-ligation of the 9 input module plasmids. M: GeneRuler 1kb DNA Ladder Plus from Fermentas. Restriction-ligation was performed at 37°C for 3 (lane 3h) or 6 hours (lane 6h) or with 25 cycles (2 min 37°C+5 min 16°C, lane 25) or 50 cycles (lane 50), and without BsaI enzyme (lane nb). The arrow indicates the 1.17 kb linear assembled GFP gene product. (C) Ethidium bromide-stained gels of 72 minipreps digested with XmaI and AvrII (expected fragment sizes: 4.6 kb, 945 and 555 bp), obtained from restriction-ligations performed for 6 h 37°C (6 h), for 25 or 50 cycles (25×/50×), with normal ligase (nl) or high concentration ligase (hcl). Numbers indicate minipreps with an incorrect restriction pattern, and stars indicate constructs that consist of dimers (same restriction pattern as monomers). V, vector pX-lacZ.

Article Snippet: A restriction-ligation was set up by adding into a single tube 50 ng of each of the 27 trypsinogen fragment constructs , 50 ng of vector, 10 units of BsaI enzyme (NEB) and 3 units of T4 DNA ligase (Promega) in a total volume of 15 microliters in ligation buffer (Promega).

Techniques: Construct, Plasmid Preparation, Staining, Ligation, Concentration Assay

Journal: PLoS ONE

Article Title: Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

doi: 10.1371/journal.pone.0005553

Figure Lengend Snippet: DNA shuffling strategy. (A) Two DNA ends terminated by the same 4 nucleotides (sequence f, composed of nucleotides 1234, complementary nucleotides noted in italics) flanked by a BsaI recognition sequence, B, form two complementary DNA overhangs after digestion with BsaI. (B) For shuffling, genes of interest are aligned, and recombination points consisting of 4 nucleotide sequences (f1 to fn+1) are defined on conserved sequences. Module fragments (core sequence, C1 to Cn, plus flanking 4 nucleotide sequences) are amplified by PCR and cloned in an intermediate cloning vector. Module fragment plasmids and the acceptor vector are assembled in one restriction-ligation with BsaI and ligase. S1 and S2, two different selectable markers. Z, lacZ alpha gene fragment.

Article Snippet: A restriction-ligation was set up by adding into a single tube 50 ng of each of the 27 trypsinogen fragment constructs , 50 ng of vector, 10 units of BsaI enzyme (NEB) and 3 units of T4 DNA ligase (Promega) in a total volume of 15 microliters in ligation buffer (Promega).

Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Ligation

Journal: PLoS ONE

Article Title: Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

doi: 10.1371/journal.pone.0005553

Figure Lengend Snippet: Shuffling of trypsinogen. (A) Alignment of the aminoacid sequence of bovine cationic trypsinogen (BC), bovine anionic trypsinogen (BA) and human cationic trypsinogen (HC). Nucleotide sequence of the chosen recombination sites is shown. (B) Map of the 27 trypsinogen module plasmids, the acceptor vector, and of an example of one of the resulting shuffled construct obtained. B, BsaI restriction site. S, K: spectinomycin and kanamycin resistance genes. RB/LB, T-DNA right and left borders. AttB, Phage C31 recombination site, N tobamoviral 3′ non-translated region, T, Nos terminator. (C) Ethidium bromide-stained gels of 28 minipreps prepared from single colonies (1 to 24) or from 4 libraries (L1–4, approximately 700 clones in each) digested with XmaI (incorrect pattern 1, 2, 7 and 17).

Article Snippet: A restriction-ligation was set up by adding into a single tube 50 ng of each of the 27 trypsinogen fragment constructs , 50 ng of vector, 10 units of BsaI enzyme (NEB) and 3 units of T4 DNA ligase (Promega) in a total volume of 15 microliters in ligation buffer (Promega).

Techniques: Sequencing, Plasmid Preparation, Construct, Staining, Clone Assay