bcli  (New England Biolabs)

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: Current protocols in molecular biology

Article Title: Simple CRISPR-Cas9 Genome Editing in S. cerevisiae

doi: 10.1002/cpmb.110

Figure Lengend Snippet: Experimental strategy for cloning user-designed guide sequence into sgRNA expression cassette in pML104 or pML107. The BclI site is located at the 3’ end of the pSNR52 promoter, which is used to drive sgRNA expression in yeast. The SwaI site is located in the structural portion of the sgRNA. SwaI and BclI ).

Article Snippet: Restriction enzymes SwaI (NEB #R0604S) and BclI (NEB #R0160S).

Techniques: Clone Assay, Sequencing, Expressing

Journal: Current protocols in molecular biology

Article Title: Simple CRISPR-Cas9 Genome Editing in S. cerevisiae

doi: 10.1002/cpmb.110

Figure Lengend Snippet: Plasmid maps of the sgRNA/Cas9 expression vectors pML104 ( URA3 marker) and pML107 ( LEU2 marker). Both plasmids are yeast/ E. coli shuttle vectors containing an ampicillin resistance (AmpR) marker, and a yeast 2 micron (2μ) origin of replication. Both vectors contain a Cas9 expression cassette and an sgRNA expression cassette, which contains unique BclI and SwaI restriction sites, to facilitate directional cloning of the user-designed guide sequence into the sgRNA expression cassette.

Article Snippet: Restriction enzymes SwaI (NEB #R0604S) and BclI (NEB #R0160S).

Techniques: Plasmid Preparation, Expressing, Marker, Clone Assay, Sequencing

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