Journal: Methods in enzymology

Article Title: Reconstituting the 4-Strand DNA Strand Exchange

doi: 10.1016/bs.mie.2017.11.013

Figure Lengend Snippet: The scheme of 4-strand exchange reaction promoted by hRad51 and E. coli RecA. (A) Joint molecules with the 3′-ssDNA displaced strand are produced by hRad51 using gapped DNA and XhoI-linearized pBS II SK(+) dsDNA. (B) Joint molecules with the 5′-ssDNA displaced strand are produced by RecA using gapped DNA and AlwNI-linearized pBS II SK(+) dsDNA. Curved arrows show the direction of polymerization of hRad51 (3′ → 5′) and RecA (5′ → 3′) on the ssDNA region of gapped DNA to initiate the reactions.

Article Snippet: Alw NI generates DNA ends with 3′ overhangs, which may be 3′ radio-labeled with terminal deoxynucleotidyl transferase. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Mix in 100 μL reaction 10 μg of pBS II SK (+) plasmid DNA, 10 μL of 10 × NEBuffer 4, and 4 μL of Alw NI (10 units/μL).

Techniques: Produced

Journal: Methods in enzymology

Article Title: Reconstituting the 4-Strand DNA Strand Exchange

doi: 10.1016/bs.mie.2017.11.013

Figure Lengend Snippet: Construction of gapped DNA. (i) pBS II SK(+) plasmid DNA is cleaved with XhoI and AlwNI restriction endonucleases. (ii) The 2065 bp XhoI–AlwNI dsDNA fragment is purified by electrophoresis in agarose gels, then (iii) denatured and annealed to circular ssDNA to produce gapped DNA. (iv) Finally, the gapped DNA is purified by electrophoresis in agarose gels.

Article Snippet: Alw NI generates DNA ends with 3′ overhangs, which may be 3′ radio-labeled with terminal deoxynucleotidyl transferase. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Mix in 100 μL reaction 10 μg of pBS II SK (+) plasmid DNA, 10 μL of 10 × NEBuffer 4, and 4 μL of Alw NI (10 units/μL).

Techniques: Plasmid Preparation, Purification, Electrophoresis

Journal: Methods in enzymology

Article Title: Reconstituting the 4-Strand DNA Strand Exchange

doi: 10.1016/bs.mie.2017.11.013

Figure Lengend Snippet: Purification of the 2065 bp XhoI–AlwNI dsDNA fragment of pBS II SK(+) (A) and the gapped DNA (B) by electrophoresis in agarose gels. (A) After electrophoresis, lanes A, C, and E are excised from the gel and stained with ethidium bromide to visualize the DNA bands. Using these bands as markers, the area of the gel with the 2065 bp dsDNA fragment (A) and with the gapped DNA (B) is cut out of lanes B and D (dashed white boxes), and then the DNA is extracted from the gel by electroelution.

Article Snippet: Alw NI generates DNA ends with 3′ overhangs, which may be 3′ radio-labeled with terminal deoxynucleotidyl transferase. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Mix in 100 μL reaction 10 μg of pBS II SK (+) plasmid DNA, 10 μL of 10 × NEBuffer 4, and 4 μL of Alw NI (10 units/μL).

Techniques: Purification, Electrophoresis, Staining

Journal: Methods in enzymology

Article Title: Reconstituting the 4-Strand DNA Strand Exchange

doi: 10.1016/bs.mie.2017.11.013

Figure Lengend Snippet: Analysis of the DNA substrates, intermediates, and gapped DNA product by electrophoresis in a 1.4% agarose gel. Lane 1: 1-kb DNA ladder. Lane 2, 3, and 5: purified circular ssDNA pBS II SK (+) (100 ng), 2065 bp dsDNA (125 ng), and gapped DNA (125 ng), respectively. Lane 4: DNA products of the annealing reaction (415 ng).

Article Snippet: Alw NI generates DNA ends with 3′ overhangs, which may be 3′ radio-labeled with terminal deoxynucleotidyl transferase. list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Mix in 100 μL reaction 10 μg of pBS II SK (+) plasmid DNA, 10 μL of 10 × NEBuffer 4, and 4 μL of Alw NI (10 units/μL).

Techniques: Electrophoresis, Agarose Gel Electrophoresis, Purification