bcli  (New England Biolabs)

Journal: PLoS ONE

Article Title: Identification of Rv3852 as an Agrimophol-Binding Protein in Mycobacterium tuberculosis

doi: 10.1371/journal.pone.0126211

Figure Lengend Snippet: Construction of rv3852 knockout Mtb and verification by Southern blot and PCR. (A) Upper panel displays the genetic organization of the rv3852 region in Mtb (WT), Lower panel displays the same region with replacement of rv3852 by hygromycin resistance gene in rv3852 knockout Mtb (KO). Filled rectangle indicates the location of probe used in the Southern blot. Sites of digestion by BclI on genomic DNA from WT and KO as well as the sizes of the generated DNA fragment (a and b) are demonstrated under each panel. PCR products from genomic DNA from WT (1 and 2) and KO (3 and 4) are denoted. (B) Left, Southern blot of BclI digested genomic DNA from WT and KO. Calculated sizes of the fragments hybridizing with the probe were 3714 bp (WT) and 2916 bp (KO) as indicated in A. Right, PCR products from genomic DNA from WT and KO. The calculated sizes of the PCR products were 902 (Lane 1), 860 (Lane 2) for WT and 1322 (Lane 3), 1560 bp (Lane 4) for KO as indicated in A.

Article Snippet: For Southern blot analysis, genomic DNA from wild type Mtb and hygromycin resistant Mtb were digested by BclI (New England BioLabs) at 50°C overnight, run on the 1% agarose gel, transferred to the membrane (Amersham) and blotted by a designed probe.

Techniques: Knock-Out, Southern Blot, Polymerase Chain Reaction, Generated

Journal: The Journal of Experimental Medicine

Article Title: Herpesvirus telomeric repeats facilitate genomic integration into host telomeres and mobilization of viral DNA during reactivation

doi: 10.1084/jem.20101402

Figure Lengend Snippet: Integration does not occur in host telomeres in the absence of viral TMRs. (A) Schematic representation and corresponding PFGE and Southern blot analysis of LCL DNA digested with SfiI. Fragment sizes generated by SfiI digestion of integrated and nonintegrated MDV genomes are depicted and sizes are given. The size of the linear MDV genome observed during lytic replication is indicated by an arrow. Results are representative of three independent Southern blot analyses. (B) Southern blotting of DNA of LCL derived from animals infected with vTE1 and digested with BclI. Potential intragenomic and telomeric integration sites are indicated. Results are representative of three independent Southern blot analyses. (C) Quantification of MDV copies in tumor cells. Results are shown as mean herpesvirus genome copies detected by the TR L probe relative to B2M in three independent experiments. The data are shown relative to LCL CU482 derived from a vRB-1B–infected chicken with standard deviations (error bars).

Article Snippet: 1 × 107 /ml LCLs were embedded in 1% agarose and digested at 50°C for 48 h hours in lysis buffer (0.5 M EDTA and 1% wt/vol N -laurylsarcosine) containing 1 mg/ml proteinase K. Proteinase K was inactivated with 0.01 mM phenylmethanesulfonyl fluoride, and agarose plugs were digested with either SfiI or BclI (New England Biolabs) overnight according to the manufacturer’s instructions.

Techniques: Southern Blot, Generated, Derivative Assay, Infection

Journal: The Journal of Experimental Medicine

Article Title: Attracting AID to targets of somatic hypermutation

doi: 10.1084/jem.20090821

Figure Lengend Snippet: DNase I hypersensitive sites of GFP transgenes. (A) Southern blot of two C-GFP and two A-GFP subclones. DNA from DNase I–digested cells was digested with Pst I and Bcl I and hybridized with a GFP probe. 0, 10, and 50 U DNase I are indicated by the filled horizontal arrows. (B) Ethidium bromide–stained gel of DNA fragments before hybridization for A. (C) An ovalbumin probe was used to reprobe the blot in A. (D) Vertical arrows indicate DNase I hypersensitive sites observed with GFP for C-GFP and A-GFP clones. The horizontal blue box indicates the probe. The experiment was repeated with DNase I digestion of different C-GFP and A-GFP cell clones showing no difference in DNase hypersensitivity.

Article Snippet: Isolated DNA was digested with Pst I and Bcl I (New England Biolabs, Inc.), and a Sac I/BsrG I fragment from eGFP was used to make a probe using a random-priming synthesis kit (Roche) for Southern blot analysis.

Techniques: Southern Blot, Staining, Hybridization, Clone Assay

Journal: mBio

Article Title: Genetic Stabilization of the Drug-Resistant PMEN1 Pneumococcus Lineage by Its Distinctive DpnIII Restriction-Modification System

doi: 10.1128/mBio.00173-15

Figure Lengend Snippet: Characterization of (R-M system) DpnIII demonstrating that R.DpnIII cleaves DNA at 5′ GATC 3′ and M.DpnIII methylates DNA at the cytosine. (A) Digestion of pUC19 and spectinomycin R with a histidine-tagged DpnIII-enriched fraction and Sau3AI, showing bands consistent with digestion at GATC. (B) Genomic DNA isolated from the WT and RMKO strains combined with endonucleases that cleave at GATC but are inhibited by methylation at different positions (cleavage by BamHI, BglII, and Sau3AI is inhibited by methylation of the cytosine, and cleavage by BclI and MboI is inhibited by methylation of the adenine). (C) WT and RMKO DNA mixed with Sau3AI and histidine-tagged DpnIII, where only the RMKO is susceptible to digestion. Further, WT DNA of strain 8140 is protected by digestion with Sau3AI and DpnIII. Enz., enzyme; MM, mass markers. The values to the left of panel A are molecular masses in base pairs.

Article Snippet: The DNA methylation status of SV35-T23 and SV35-RMKO was determined by digesting 800 ng of genomic DNA with BamHI, BclI, BglII, MboI, or Sau3AI from New England Biolabs for 2 h according to the manufacturer’s instructions, and digested DNAs were visualized on a 1% agarose gel.

Techniques: Isolation, Methylation