Assembly and Micromanipulation of Xenopus In Vitro–Assembled Mitotic Chromosomes
Mitotic chromosomes have fascinated scientists for several decades. Despite the numerous microscopy studies, chromosome structure is, however, still poorly understood. This is due to both the high complexity of the mitotic chromosomes and the lack of other appropriate techniques suitable for studying their organization. This unit describes a novel physical approach based on measurements of mitotic chromosome elasticity. The elasticity properties are determined by the underlying structure, and knowledge of them has allowed a description of the organization of the mitotic chromosomes and critical analysis of the available models. In this unit, a detailed protocol for the measurements of the elastic response of in vitro assembled mitotic chromosomes in Xenopus egg extract is presented.
Keywords: Chromosome; Xenopus; Micromanipulation; Elasticity
Table of Contents
- Basic Protocol 1: Stretching a Mitotic Xenopus Chromosome
- Support Protocol 1: Preparing Xenopus Sperm Nuclei
- Support Protocol 2: Assembling Xenopus Chromosomes
- Support Protocol 3: Building the Experimental Setup for Measurement of Chromosome Elasticity
- Support Protocol 4: Designing the Micropipets
- Support Protocol 5: Calibrating Pixel Size
- Support Protocol 6: Measuring the Spring Constant of the Micropipet
- Support Protocol 7: Preparation of the Force Calibration Reference
- Reagents and Solutions
- Literature Cited
Basic Protocol 1: Stretching a Mitotic Xenopus Chromosome
Support Protocol 1: Preparing Xenopus Sperm Nuclei
Support Protocol 2: Assembling Xenopus Chromosomes
Support Protocol 3: Building the Experimental Setup for Measurement of Chromosome Elasticity
Support Protocol 4: Designing the Micropipets
Support Protocol 5: Calibrating Pixel Size
Support Protocol 6: Measuring the Spring Constant of the Micropipet
Support Protocol 7: Preparation of the Force Calibration Reference
Figure 22.9.3 Positions of the two micropipets and the chromosome during a stretching experiment. The two micropipets are positioned upon on the y‐axis, and each one has caught one end of the chromosome.
Figure 22.9.5 Diagram of the reservoir used for chromosome stretching experiments. An O‐ring is attached to a coverslip with adhesive and the chromosome suspension is poured into the cavity formed by the O‐ring.
Figure 22.9.6 Correct geometry of the filament of the micropipet puller. The filament must have a trapezoidal shape with the length of the smallest base being 2/3 of the length of the largest base.
Figure 22.9.7 Size calibration of the pixel. The graduations of the stage micrometer are 10 µm apart. In order to calibrate the size of the pixel on the image it is necessary to measure the number of pixels between graduations.
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