scientificprotocols

Authors: Kayoko Hayashihara , Susumu Uchiyama , Shouhei Kobayashi , Masanobu Yanagisawa , Sachihiro Matsunaga & Kiichi Fukui 

Introduction

Given that DNA on which genomic information is written exists as chromosomes in a cell, handling chromosomes in vitro as experimental materials can provide varieties of information throughout life sciences. Metaphase chromosomes are highly delicate under in vitro conditions, moreover, it has been difficult to prepare massive chromosomes as experimental materials. These inconvenient points have prevented researchers to use chromosomes as the materials for in vitro experiments, although numerous microscopic observations have been so far performed. There is a standard protocol to prepare mitotic metaphase chromosomes, i.e., PA method (1, 2, 5-7). However the chromosomes prepared by the method have been found to contain lots of contaminated proteins (4). Ordinary purification processes, i.e., the sucrose density gradient centrifugation (4) often or usually result in

scientificprotocols

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scientificprotocols

Authors: Ebbe Andersen & Morten Nielsen

Introduction

Structural DNA nanotechnology has been heavily dependent on the development of dedicated software tools for the design of unique helical junctions, to define unique sticky-ends for tile assembly, and for predicting the products of the self-assembly reaction of multiple DNA strands [1-3]. Recently, several dedicated 3D editors for computer-aided design of DNA structures have been developed [4-7]. However, many of these tools are not efficient for designing DNA origami structures that requires the design of more than 200 unique DNA strands to be folded along a scaffold strand into a defined 3D shape [8]. We have recently developed a semi-automated DNA origami software package [9] that uses a 2D sequence editor in conjunction with several automated tools to facilitate the design process. Here we extend the use of the program for designing DNA origami structures in 3D and show the application by the construction of a DNA box with dimensions of 42×36

scientificprotocols

Authors: Spencer Reisbick & Patrick Willoughby

Abstract

This protocol describes an approach to preparing a series of Gaussian 09 computational input files for an ensemble of conformers generated in Spartan’14. The resulting input files are necessary for computing optimum geometries, relative conformer energies, and NMR shielding tensors using Gaussian. Using the conformational search feature within Spartan’14, an ensemble of conformational isomers was obtained. To convert the structures into a format that is readable by Gaussian 09, the conformers were first exported to a single “.sdf” file. A Python script was used to (i) read the structural information of each conformer within the “.sdf” file and (ii) write the corresponding atomic coordinates into a series Gaussian 09 input files. This approach decreases the amount of active effort required to compute NMR chemical shifts of a structure that populates an ensemble of conformers.

Introduction

NMR spectroscopy is the most useful tool for deter

scientificprotocols

Typically, an equal volume of TE-saturated phenol is added to an aqueous DNA sample in a microcentrifuge tube. The mixture is vigorously vortexed, and then centrifuged to enact phase separation. The upper, aqueous layer carefully is removed to a new tube, avoiding the phenol interface and then is subjected to two ether extractions to remove residual phenol. An equal volume of water-saturated ether is added to the tube, the mixture is vortexed, and the tube is centrifuged to allow phase separation. The upper, ether layer is removed and discarded, including phenol droplets at the interface. After this extraction is repeated, the DNA is concentrated by ethanol precipitation.

Protocol

1. Add an equal volume of TE-saturated phenol to the DNA sample contained in a 1.5 ml microcentrifuge tube and vortex for 15-30 seconds.

• Centrifuge the sample for 5 minutes at room temperature to separate the phases.
• Remove about 90% of the upper, aqueous layer to a clean tube, carefully avoiding proteins at the aqueous:ph

scientificprotocols

Authors: Giorgio Pochetti & Roberta Montanari

Abstract

It could be very useful in drug development to determine the relative affinities of a ligand that can bind to a protein with two (or more) different and specific binding sites. X-ray crystallography is a very powerful technique to determine the locations and describe the features of each binding site but it is unuseful to establish their relative affinities. Moreover, the stoichiometry of binding observed in the crystal structure of a complex could be an artifact of the crystallization conditions.

On the contrary, isothermal titration calorimetry (ITC) can be used to assign the correct stoichiometry of the association, depending on the initial and final concentration ratio between ligand and protein, and to establish the affinity for each specific binding site, if combined with structural information from X-ray crystallography.

scientificprotocols

Authors: Manuel Théry and Matthieu Piel

Corresponding authors (manuel.thery@cea.fr; matthieu.piel@curie.fr)

INTRODUCTION

This protocol describes a simple, fast, and efficient method for making adhesive micropatterns that can be used to control individual cell shape and adhesion patterns. It is based on the use of an elastomeric stamp containing microfeatures to print proteins on the substrate of choice. The process can be subdivided into three parts. First, a silicon master is fabricated, which contains the microfeatures of interest. Once fabricated, the master can be used multiple times to make stamps. Masters with customized patterns can also be purchased commercially. Second, a polydimethylsiloxane (PDMS) stamp is fabricated. Unlike fabrication of the master, this step can be performed without specialized equipment. The PDMS stamp is inked with extracellular matrix proteins. Proteins are printed on a substrate (e.g., a tissue culture polystyrene d

scientificprotocols

Authors: Dong-sheng Li, Jianqiang Hao, Ya-Hong Yuan, Se Hak Yun, Jing-Bo Feng, Long-Jun Dai & Garth L. Warnock

Abstract

Mouse islet subcapsule transplantation is widely used in diabetes-related studies. Reliable and reproducible transplantation is essential to the success of those types of investigations. The present protocol presents detailed procedures on the mouse diabetic model and islet implantation. The blood glucose tracings under varied transplant circumstances are presented, covering syngeneic, allogeneic and xenogeneic islet transplantations. This protocol is straightforward and has been proven to be practical and reproducible.

Introduction

Islet transplantation has become a very progressive field in diabetes studies in the recent few decades. It took almost 30 years from the first islet transplantation in rats to the first successful islet allotransplantation in patients with type 1 diabetes 1,2. Islet transplantation as an important procedure is widely used in almost all diabetes-

scientificprotocols

Authors: Rachel Wood, Angelique Luabeya, Kris Weigel, Alicia Wilbur, Lisa Jones-Engel, Mark Hatherill & Gerard Cangelosi

Abstract

Diagnosis of pulmonary tuberculosis (TB) usually includes laboratory analysis of sputum, a viscous material derived from deep in the airways of patients with active disease. As a diagnostic sample matrix, sputum can be difficult to collect and analyze. An alternative, less invasive sample matrix could greatly simplify TB diagnosis. We hypothesized that Mycobacterium tuberculosis cells or DNA accumulate on the oral epithelia of pulmonary TB patients, and can be collected and detected by using oral (buccal) swabs combined with PCR detection of M. tuberculosis DNA. A case-control study (Wood RC et al, SREP-14-09269B, 2015) supported the efficacy of this approach. Oral swab samples are non-invasive, non-viscous, and easy to collect with or without active TB symptoms.

Procedure

A. Prepare for buccal swab collection

scientificprotocols

Authors: Yalin Tang, Qian Shang, Junfeng Xiang, Qianfan Yang, Qiuju Zhou, Lin Li, Hong Zhang, Qian Li, Hongxia Sun, Aijiao Guan, Wei Jiang & Wei Gai

Abstract

This protocol presents the screening of ligand(s) from medicinal plant extracts based on target recognition by using NMR spectroscopy. A detailed description of sample preparation and analysis process is provided. NMR spectroscopies described here are 1H NMR, diffusion-ordered spectroscopy (DOSY), relaxation-edited NMR, 1H–13C HSQC and HMBC. This method includes three steps: First, investigate the NMR spectroscopy properties of target and choose the suitable spectral editing NMR method; second, judge the existence of ligand(s) and determine the proton signals of ligand(s) in the extracts; third, verify the structure(s) of the ligand(s). This method allows the direct structural identification of ligand(s) from medicinal plant extracts without separation and purification. Thus it provides a very promising strategy for the fast screening of lead