Help with understanding this section from a research paper. My instructor wants

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Help with understanding this section from a research paper. My instructor wants us to summarize the materials and methods. It's kinda hard to do when I have no clue what it's saying. So if someone can better explain it that be great! DNA curvature prediction and electrophoretic mobility shift assays: The upaC promoter region was analyzed in silico using bend.it, a program that enables the prediction of a curvature propensity plot calcu- lated with DNase I-based parameters . The curvature is calculated as a vector sum of dinucleotide geome- tries (roll, tilt, and twist angles) and expressed as degrees per helical turn (10.5°/helical turn 1°/bp). Experimentally tested curved motifs produce curvature values of 5 to 25°/helical turn, whereas straight motifs give val- ues below 5°/helical turn. The upaC 250-bp promoter region was ampli-fied using primers upaC.pro.ext-5 250 and upaC.pro.ext-3-1ATG, and its intrinsic curvature was assessed by comparing its electrophoretic mo- bility with that of an unbent marker fragment (Promega; 100-bp DNA ladder) on a 0.5% Tris-borate-EDTA (TBE), 7.5% PAGE gel at 4°C for retarded gel electrophoretic mobility. Gel shift assays were performed essentially as previously described (3). A DNA mixture comprising an equimolar ratio of the PCR-amplified upaC promoter region and TaqI-SspI-digested pBR322 was incubated at room temperature for 15 min with increasing amounts of native purified H-NS protein (a gift from S. Rimsky) in 30 l of reaction mixture con- taining 40 mM HEPES (pH 8), 60 mM potassium glutamate, 8 mM mag- nesium aspartate, 5 mM dithiothreitol, 10% glycerol, 0.1% octylphenoxy- polyethoxyethanol, 0.1 mg/ml BSA (H-NS binding buffer). DNA fragments and DNA-protein complexes were resolved by gel electropho- resis (0.5% TBE, 3% MS agarose gel run at 50 V at 4°C) and visualized after staining with ethidium bromide. Help with understanding this section from a research paper. My instructor wants us to summarize the materials and methods. It's kinda hard to do when I have no clue what it's saying. So if someone can better explain it that be great! DNA curvature prediction and electrophoretic mobility shift assays: The upaC promoter region was analyzed in silico using bend.it, a program that enables the prediction of a curvature propensity plot calcu- lated with DNase I-based parameters . The curvature is calculated as a vector sum of dinucleotide geome- tries (roll, tilt, and twist angles) and expressed as degrees per helical turn (10.5°/helical turn 1°/bp). Experimentally tested curved motifs produce curvature values of 5 to 25°/helical turn, whereas straight motifs give val- ues below 5°/helical turn. The upaC 250-bp promoter region was ampli-fied using primers upaC.pro.ext-5 250 and upaC.pro.ext-3-1ATG, and its intrinsic curvature was assessed by comparing its electrophoretic mo- bility with that of an unbent marker fragment (Promega; 100-bp DNA ladder) on a 0.5% Tris-borate-EDTA (TBE), 7.5% PAGE gel at 4°C for retarded gel electrophoretic mobility. Gel shift assays were performed essentially as previously described (3). A DNA mixture comprising an equimolar ratio of the PCR-amplified upaC promoter region and TaqI-SspI-digested pBR322 was incubated at room temperature for 15 min with increasing amounts of native purified H-NS protein (a gift from S. Rimsky) in 30 l of reaction mixture con- taining 40 mM HEPES (pH 8), 60 mM potassium glutamate, 8 mM mag- nesium aspartate, 5 mM dithiothreitol, 10% glycerol, 0.1% octylphenoxy- polyethoxyethanol, 0.1 mg/ml BSA (H-NS binding buffer). DNA fragments and DNA-protein complexes were resolved by gel electropho- resis (0.5% TBE, 3% MS agarose gel run at 50 V at 4°C) and visualized after staining with ethidium bromide. DNA curvature prediction and electrophoretic mobility shift assays: The upaC promoter region was analyzed in silico using bend.it, a program that enables the prediction of a curvature propensity plot calcu- lated with DNase I-based parameters . The curvature is calculated as a vector sum of dinucleotide geome- tries (roll, tilt, and twist angles) and expressed as degrees per helical turn (10.5°/helical turn 1°/bp). Experimentally tested curved motifs produce curvature values of 5 to 25°/helical turn, whereas straight motifs give val- ues below 5°/helical turn. The upaC 250-bp promoter region was ampli-fied using primers upaC.pro.ext-5 250 and upaC.pro.ext-3-1ATG, and its intrinsic curvature was assessed by comparing its electrophoretic mo- bility with that of an unbent marker fragment (Promega; 100-bp DNA ladder) on a 0.5% Tris-borate-EDTA (TBE), 7.5% PAGE gel at 4°C for retarded gel electrophoretic mobility. Gel shift assays were performed essentially as previously described (3). A DNA mixture comprising an equimolar ratio of the PCR-amplified upaC promoter region and TaqI-SspI-digested pBR322 was incubated at room temperature for 15 min with increasing amounts of native purified H-NS protein (a gift from S. Rimsky) in 30 l of reaction mixture con- taining 40 mM HEPES (pH 8), 60 mM potassium glutamate, 8 mM mag- nesium aspartate, 5 mM dithiothreitol, 10% glycerol, 0.1% octylphenoxy- polyethoxyethanol, 0.1 mg/ml BSA (H-NS binding buffer). DNA fragments and DNA-protein complexes were resolved by gel electropho- resis (0.5% TBE, 3% MS agarose gel run at 50 V at 4°C) and visualized after staining with ethidium bromide. DNA curvature prediction and electrophoretic mobility shift assays: The upaC promoter region was analyzed in silico using bend.it, a program that enables the prediction of a curvature propensity plot calcu- lated with DNase I-based parameters . The curvature is calculated as a vector sum of dinucleotide geome- tries (roll, tilt, and twist angles) and expressed as degrees per helical turn (10.5°/helical turn 1°/bp). Experimentally tested curved motifs produce curvature values of 5 to 25°/helical turn, whereas straight motifs give val- ues below 5°/helical turn. The upaC 250-bp promoter region was ampli-fied using primers upaC.pro.ext-5 250 and upaC.pro.ext-3-1ATG, and its intrinsic curvature was assessed by comparing its electrophoretic mo- bility with that of an unbent marker fragment (Promega; 100-bp DNA ladder) on a 0.5% Tris-borate-EDTA (TBE), 7.5% PAGE gel at 4°C for retarded gel electrophoretic mobility. Gel shift assays were performed essentially as previously described (3). A DNA mixture comprising an equimolar ratio of the PCR-amplified upaC promoter region and TaqI-SspI-digested pBR322 was incubated at room temperature for 15 min with increasing amounts of native purified H-NS protein (a gift from S. Rimsky) in 30 l of reaction mixture con- taining 40 mM HEPES (pH 8), 60 mM potassium glutamate, 8 mM mag- nesium aspartate, 5 mM dithiothreitol, 10% glycerol, 0.1% octylphenoxy- polyethoxyethanol, 0.1 mg/ml BSA (H-NS binding buffer). DNA fragments and DNA-protein complexes were resolved by gel electropho- resis (0.5% TBE, 3% MS agarose gel run at 50 V at 4°C) and visualized after staining with ethidium bromide.

Explanation / Answer

The bend.it server calculates the curvature of DNA molecules as predicted from the DNA sequences. The calculation is based on values tabulated for dinucleotides and trinucleotides, and the curvature (degree per helical turn) is calculated using standard algorithms. Calculation was originally based on DNA bendability parameters derived from DNAseI digestion that characterise the (static or dynamic) bending of trinucleotides towards the major groove and the results can be visualised as 1D or 2D plots on the screen.

The electrophoretic mobility shift assay (EMSA) is based on the observation that protein:DNA complexes migrate more slowly than free linear DNA fragments when subjected to non-denaturing polyacrylamide or agarose gel electrophoresis. Because the rate of DNA migration is shifted or retarded when bound to protein, the assay is also referred to as a gel shift or gel retardation assay.

the protein:DNA complexes(PCR-amplified upaC promoter region and TaqI-SspI-digested pBR322 with native purified H-NS protein) are quickly resolved as compared to free DNA, providing a "snapshot" of the equilibrium between bound and free DNA in the original sample. The 3 % gel matrix provides a "caging" effect that helps to stabilize the interaction complexes: even if the components of the interaction complex dissociate, their localized concentrations remain high, promoting prompt reassociation. Additionally the relatively low ionic strength of the electrophoresis buffer helps to stabilized transient interactions, permitting even labile complexes to be resolved and analyzed by this method..

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