Protocol for the Preparation of Arabidopsis Meiotic Chromosome Spreads and Fluorescent in situ Hybridization
拟南芥减数分裂染色体的分离制备方法和荧光原位杂交技术
发布: 2014年04月20日第4卷第8期 DOI: 10.21769/BioProtoc.1102 浏览次数: 16404
评审: Renate Weizbauer
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参见作者原研究论文
The authors used this protocol in:
Sep 2013
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Abstract
This protocol is a more detailed version of previous protocols (Yang et al., 2011; Bolaños-Villegas et al., 2013) developed for the examination of meiotic chromosome spreads. Meiotic chromosome spreads are useful to determine the presence of defects in chromosome pairing and segregation. The protocol also describes how to perform fluorescent in situ hybridization experiments with a centromere probe used to label chromosomes.
Keywords: Meiocyte (性细胞)
Cell cycle (细胞周期)
Centromere (着丝粒)
Reproduction (繁殖)
Higher plants (高等植物)
Materials and Reagents
- Inflorescences from young, healthy Arabidopsis thaliana plants
- Agar plates, with 1/2x Murashige and Skoog solid medium
- Pectolyase (Sigma-Aldrich, catalog number: P3026 )
- Cellulase (Sigma-Aldrich, catalog number: C1184 )
- β-glucoronidase (Roche Diagnostics, catalog number: 03707598001 )
- Sucrose (Affymetrix, catalog number: 21938 )
- Ethanol (EMD Millipore, catalog number: 1085430250 )
- Chloroform (EMD Millipore, catalog number: 102442 )
- Glacial acetic acid (EMD Millipore, catalog number: 100063 )
- Citric acid anhydrous (Affymetrix, catalog number: 13729 )
- Sodium citrate dihydrate (Affymetrix, catalog number: 13735 )
- Formamide (Sigma-Aldrich, catalog number: F9037 )
- Saline-sodium citrate (SSC) (Sigma-Aldrich, catalog number: S0902 )
- Dextran sulfate (Sigma-Aldrich, catalog number: 42867 )
- Salmon sperm (Life Technologies, catalog number: 15632-011 )
- VECTASHIELD® mounting medium with DAPI (10 ml) (Vector Laboratories, catalog number: H-1400 )
- Fluorescein High-Prime labeling kit (Roche Diagnostics, catalog number: 11585622910 )
- pAL1 centromere probe (ABRC, catalog number: CD3-16 )
- Taq DNA polymerase with standard Taq buffer (New England Biolabs, catalog number: M0273S )
- QIAquick Gel Extraction Kit (kit for purification of DNA from gels) (QIAGEN, catalog number: 28704 )
- Gel Pilot 1 Kb Ladder (molecular weight marker for DNA) (QIAGEN, catalog number: 239085 )
- Agarose for electrophoresis (Bio-Rad Laboratories, catalog number: BR 161-3100 )
- Carnoy’s solution (see Recipes)
- 10 mM Sodium citrate buffer (see Recipes)
- Enzyme digestion buffer (see Recipes)
- FISH buffer (see Recipes)
- 10x PBS (see Recipes)
- 20x SSC (see Recipes)
Equipment
- Hypodermic needles and syringe (Terumo Medical Corporation, model: 26G x ½”, catalog number: NN-2613R )
- Tweezers (Dumont)
- Poly-Prep® poly-L-lysine coated slides (Sigma-Aldrich, catalog number: P0425-72EA )
- Nunc® Lab-Tek® II slides (Thermo Fisher Scientific, catalog number: 154453 )
- Coverslips (Matsunami Glass, catalog number: C218181 )
- Hybridisation oven OV1 (Biometra, catalog number: 052-090 )
- Thermal cycler (Biometra, catalog number: 050-551 )
- Dry block (Thermo Fisher Scientific, Reacti-Therm®)
- -86 °C refrigerator (Thermo Fisher Scientific, model: Forma® 88000 )
- Frigidaire -20 °C refrigerator (Frigidaire, model: FCFS201LFB )
- Fluorescence microscope
- Coplin staining jars (Thermo Fisher Scientific, catalog number: 107 )
- Rubbermaid LunchBlox® food containers
- Super Pap Pen’ liquid blocker pen (Electron Microscopy Sciences, catalog number: 71310 ) (optional)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Bolaños-Villegas, P., Yang, X., Makaroff, C. A. and Jauh, G. (2014). Protocol for the Preparation of Arabidopsis Meiotic Chromosome Spreads and Fluorescent in situ Hybridization. Bio-protocol 4(8): e1102. DOI: 10.21769/BioProtoc.1102.
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分类
植物科学 > 植物细胞生物学 > 细胞成像
细胞生物学 > 细胞结构 > 染色体
分子生物学 > DNA > DNA 结构
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