Journal: The Plant Cell

Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta

doi:

Figure Lengend Snippet: A Low-Copy Repeat Sequence Identifies Restriction Fragments Distal to the AVR-Pita Telomere That Are Deleted in Some Spontaneous Mutants. Genomic DNAs were digested with BglII for DNA gel blot analysis. Hybridization with the 10-kb XhoI fragment from cosmid A10H8 identified the 6.5-kb telomeric BglII fragment and an additional 4.0-kb fragment (arrows) that was altered in avr-pita − mutants. All avirulent laboratory strains inherited these two bands from the Chinese field isolate O-137 (lane 23) through the RFLP mapping strain 4224-7-8 (lane 2). The virulent mapping parent 6043 (lane 1) does not have these bands. Not shown are data consistent with this result but obtained by using SalI, EcoRI, and SacI. Except for lane 1, which contains DNA from the virulent parent 6043, each v lane contains DNA from a virulent mutant obtained from the first avirulent strain (A lanes) to its left. Lane 1, 6043 (v); lane 2, 4224-7-8 (A); lane 3, 4360-17-1 (A); lane 4, CP917 (v); lane 5, 4375-R-26 (A); lane 6, CP984 (v); lane 7, 4375-R-39 (A); lane 8, CP918 (v); lane 9, 4375-R-6 (A); lane 10, CP983 (v); lane 11, CP1614 (v); lane 12, CP1615 (v); lane 13, CP1631 (A); lane 14, CP1632 (v); lane 15, CP1634 (A); lane 16, CP1635 (v); lane 17, CP1637 (A); lane 18, CP1638 (v); lane 19, CP1640 (A); lane 20, CP1641 (v); lane 21, CP1643 (A); lane 22, CP1644 (v); and lane 23, O-137 (A). Markers at left identify the positions of λ HindIII DNA length standards, which are as follows (from the top): 23.1, 9.4, 6.6, 4.4, 2.3, 2.0, and 0.56 kb.

Article Snippet: Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs).

Techniques: Sequencing, Western Blot, Hybridization, Mutagenesis

Journal: BMC Biotechnology

Article Title: Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers

doi: 10.1186/1472-6750-8-23

Figure Lengend Snippet: Structure of the DNA molecules used in this study. A . Structure of the parent c-DNA. B . l-DNA formed by restriction digest of c-DNA with the enzyme Xho I. The size of the linearized plasmid was 4272 bp, C . pcr-DNA (2209 bp) generated by site-specific primers that amplified the region of the c-DNA containing the HTLV promoter, the hOPG open reading frame, and the SV40 Poly-A site. D . Structure of the parent c-DNA used in transfection. E . l-DNA formed by digest of pEGFP-N2 with the enzyme Cla I, which cuts in the vector backbone. F . pcr-DNA (1713 bp) generated by primers that are complementary to the sequence upstream of the CMV promoter and downstream of the polyA site.

Article Snippet: Linear DNA (l-DNA) Purified c-DNA was linearized using the restriction enzyme, Xho I (New England Biolabs; Pickering, ON) for pORF9-hTNFRS11b or Cla I (Invitrogen; Burlington, ON) for pEGFP-N2, Restriction digestion were set up with 5 μg of DNA per 50 μL of reaction volume containing 3 units of enzyme and incubated at 37°C for 16 hours.

Techniques: Plasmid Preparation, Polymerase Chain Reaction, Generated, Amplification, Transfection, Sequencing

Journal: Journal of Biological Engineering

Article Title: AFM Imaging Reveals Multiple Conformational States of ADAMTS13

doi: 10.1186/s13036-018-0102-y

Figure Lengend Snippet: AFM system successfully imaged proteins . ( a ) WT- and GOF-ADAMTS13 plasmids were double digested by restriction enzymes HindIII and XhoI, and verified in agarose gel electrophoresis. ( b ) Purified WT- and GOF-ADAMTS13 were analyzed by SDS-PAGE under reducing conditions on a 7.5% gel and Western blotting with anti-His antibody. ( c ) AFM images of VWF-A1 (30 kDa), BSA (67 kDa), anti-His tag antibody (150 kDa) and commercial ADAMTS13 (190 kDa). The horizontal scale bar is 200 nm. The vertical scale bar indicates the height. ( d ) The plot of volumes of these four proteins versus their molecular weights. The data was well fitted into a straight line. The goodness of fit is indicated by R 2 . According to the linear relationship y = 5.31817*x + 256.175, the volume of purified WT-ADAMTS13 (red square) corresponds to the molecular weight of 213 kDa. Data were presented as mean ± SD.

Article Snippet: Restriction enzymes XhoI and HindIII-HF were purchased from NEB (Ipswich, MA, USA).

Techniques: Agarose Gel Electrophoresis, Purification, SDS Page, Western Blot, Molecular Weight

Journal: Nucleic Acids Research

Article Title: Structure of discontinuities in kinetoplast DNA-associated minicircles during S phase in Crithidia fasciculata

doi: 10.1093/nar/gkm1061

Figure Lengend Snippet: Ligation of discontinuities in kinetoplast-associated minicircles. Purified kDNA from 300 min after release from the hydroxyurea block were either treated with E. coli DNA ligase (+) or not (−) prior to digestion with XhoI and alkaline denaturing gel analysis. The gel was transferred and hybridized as in Figure 3 to identify released H and L single strands. Marker lanes: M1, minicircle single strand linears (ssl) and single strand circles (ssc); M2, denatured covalently closed minicircles (dcc) in addition to minicircle single strand linears (ssl) and single strand circles (ssc); M3, molecular weight markers.

Article Snippet: The remainders of the cell lysates were each ethanol precipitated and resuspended in 200 μl of 0.01 M Tris pH 8, 1 mM EDTA and 22 μl of 10 × NEB 3 buffer followed by digestion for 60 min at 37°C with Mlu I and XhoI restriction enzymes (New England Biolabs) to fragment the DNA.

Techniques: Ligation, Purification, Blocking Assay, Marker, Droplet Countercurrent Chromatography, Molecular Weight

Journal: Nucleic Acids Research

Article Title: Structure of discontinuities in kinetoplast DNA-associated minicircles during S phase in Crithidia fasciculata

doi: 10.1093/nar/gkm1061

Figure Lengend Snippet: Topoisomerase decatenation of XhoI-resistant catenated minicircle molecules. Purified kDNA prepared at 300 min was digested with XhoI and one-half was additionally treated with a type II DNA topoisomerase prior to neutral gel electrophoresis and Southern blotting using a minicircle probe. Catenated minicircles are indicated by a bracket. N/G, nicked or gapped minicircle DNA; L, unit-length linear minicircle DNA; CCC, covalently closed circular minicircle DNA.

Article Snippet: The remainders of the cell lysates were each ethanol precipitated and resuspended in 200 μl of 0.01 M Tris pH 8, 1 mM EDTA and 22 μl of 10 × NEB 3 buffer followed by digestion for 60 min at 37°C with Mlu I and XhoI restriction enzymes (New England Biolabs) to fragment the DNA.

Techniques: Purification, Nucleic Acid Electrophoresis, Southern Blot, Countercurrent Chromatography

Journal: Nucleic Acids Research

Article Title: Structure of discontinuities in kinetoplast DNA-associated minicircles during S phase in Crithidia fasciculata

doi: 10.1093/nar/gkm1061

Figure Lengend Snippet: ( A ) Physical map of C. fasciculata minicircle DNA ( 36 ). The two-replication origins, oriA and oriB, are located 180° apart on the circular DNA. Specific nicks in nicked circular (form II) minicircles identified in free minicircles replicated in an isolated kinetoplast system define the initiation sites of the leading L strand and the initiation sites of the first H-strand Okazaki fragment at each origin ( 23 ). The nucleotide coordinates are indicated for the specific nicks in the H and L strands. The region from 1041 to 1240 nt has been shown to confer a static bend in the DNA ( 42 ) and is referred to as a bent helix. ( B ) Crithidia fasciculata minicircle replication schematic. Thin line, L strand; thick line, H strand; filled arrowheads, specific nicks; double arrowhead, XhoI site.

Article Snippet: The remainders of the cell lysates were each ethanol precipitated and resuspended in 200 μl of 0.01 M Tris pH 8, 1 mM EDTA and 22 μl of 10 × NEB 3 buffer followed by digestion for 60 min at 37°C with Mlu I and XhoI restriction enzymes (New England Biolabs) to fragment the DNA.

Techniques: Isolation