Journal: Nucleic Acids Research

Article Title: Torsionally constrained DNA for single-molecule assays: an efficient, ligation-free method

doi: 10.1093/nar/gkt699

Figure Lengend Snippet: Ligation-based assembly of DNA constructs for torsionally constrained assays. ( A ) Schematic of a single-molecule optical-trapping assay where the DNA is multiply labelled in both strands at both ends. Different tags [biotin (orange) and digoxigenin (cyan)] are incorporated into short DNA molecules (purple) that were ligated onto a central unlabelled DNA molecule. ( B ) An outline of the traditional method for making torsionally constrained DNA. In this method, three different DNA molecules are made by PCR, two of which are labelled with dig and biotin, respectively (Step 1). Restriction digest (Step 2) of all three DNAs facilitates the ligation necessary to assemble the final product (Step 3). Gel purification is necessary to achieve a uniform length product (Step 4).

Article Snippet: Samples were recovered by Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Columns (Bio-Rad), and further purified by QiaQuick PCR Purification Kit (Qiagen).

Techniques: Ligation, Construct, Gel Purification

Journal: Nucleic Acids Research

Article Title: Torsionally constrained DNA for single-molecule assays: an efficient, ligation-free method

doi: 10.1093/nar/gkt699

Figure Lengend Snippet: Analysis of PCR products and purification technique using non-denaturating gel electrophoresis. ( A ) An agarose gel showing intermediate and final products. Lane M: DNA ladder (1 kb+, Invitrogen); Lane 1: Biotin-labelled megaprimer; Lane 2: Dig-labelled megaprimer; Lane 3: Pre-gel purified megaprimer PCR showing the desired product and unincorporated megaprimers; Lane 4: Final PCR product (6592 bp) after gel purification. ( B ) Fractions from continuous elution electrophoresis. Lane M: DNA ladder (1 kb, NEB); Lane 1: Mixture of PCR products (before separation); Lanes 2–5: Fraction 2, 3, 10, 15 (unreacted megaprimers); Lanes 6–10: Fraction 21, 22, 23, 25, 30 (6592-bp DNA). Fractions 21–30 were combined. ( C ) Gel analysis of the pooled product from the continuous elution electrophoresis. Lane M: DNA ladder (1 kb, NEB); Lane 1: Purified and pooled DNA.

Article Snippet: Samples were recovered by Quantum Prep Freeze ‘N Squeeze DNA Gel Extraction Spin Columns (Bio-Rad), and further purified by QiaQuick PCR Purification Kit (Qiagen).

Techniques: Purification, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Gel Purification, Electrophoresis