Need help writing a summary on this material and method section of this research

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Need help writing a summary on this material and method section of this research paper. I didn't really understand what they were saying.

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. Need help writing a summary on this material and method section of this research paper. I didn't really understand what they were saying.

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. Need help writing a summary on this material and method section of this research paper. I didn't really understand what they were saying.

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

Materials: 0.5% Tris-borate-EDTA (TBE), 7.5% PAGE gel, 100-bp DNA ladder, primers.

Methods: bend.it program was used to analyze the upaC promoter region in-silico. This program enables the prediction of a curvature propensity plot calculated with DNase I based parameters. The curvature is calculated as a vector sum of dinucleotide geometries (roll, tilt, and twist angles) and expressed in terms of degrees per helical turn (10.5°/helical turn 1°/bp). Based on prior experiments, it is observed that curved motifs produce curvature values of 5 to 25°/helical turn, whereas straight motifs give values below 5°/helical turn. The upaC promoter region which is 250 bp in length was amplified using primers 3’ ATG and 5’ up to the 250th bp. 7.5% PAGE gel, 0.5% Tris-borate-EDTA (TBE) at 4°C was performed to assess the intrinsic curvature of the promoter by comparing its electrophoretic mobility with that of an unbent marker fragment (100-bp DNA ladder) for retarded gel electrophoretic mobility.

Materials: 40 mM HEPES (pH 8), 60 mM potassium glutamate, 8 mM magnesium aspartate, 5 mM dithiothreitol, 10% glycerol, 0.1% octylphenoxy- polyethoxyethanol, 0.1 mg/ml BSA, 0.5% TBE, 3% agarose gel.

Methods: the PCR amplified upaC promoter and TaqI-SspI-digested pBR322 in equimolar concentration were added to a tube. In this tube, native purified H-NS protein was added in increasing concentration and incubated at room temperature for 15 min. The reaction was carried out in H-NS binding buffer (40 mM HEPES (pH 8), 60 mM potassium glutamate, 8 mM magnesium aspartate, 5 mM dithiothreitol, 10% glycerol, 0.1% octylphenoxy- polyethoxyethanol, 0.1 mg/ml BSA) making the final volume upto 30 µl. agarose gel electrophoresis was performed to separate the DNA fragments and DNA-protein complexes on 3% agarose at 50 v at 4 °C. After electrophoresis, gel was stained with ethidium bromide to visualize the band.

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