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Acta Agron Sin ›› 2006, Vol. 32 ›› Issue (12): 1920-1923.

• RESEARCH NOTES • Previous Articles     Next Articles

High Molecular Weight DNA Preparation from Nuclei or Chromosomes of Root Meristem of Wheat

GUO Dong-Wei12,SHE Mao-Yun23,LI Lian-Cheng2,CHEN Yao-Feng1,CHEN Ming2,XU Zhao-Shi2,MA You-Zhi2*   

  1. 1 Agronomy College, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, Shaanxi; 2 Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture/ Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081; 3 Agronomy College, Xinjiang Agricultural University, Urumqi 810012, Xinjiang, China
  • Received:2006-03-10 Revised:1900-01-01 Online:2006-12-12 Published:2006-12-12
  • Contact: MA You-Zhi

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Abstract:

A simple and practical method was optimized to overcome the limitations of current method by which HMW DNA was prepared from leaf powder. Root tips of synchronized meristem were homogenized to make preparing suspension containing intact metaphase chromosomes and nuclei, which were further isolated by sucrose gradient centrifugation and flow sorting, respectively. Finally, HMW DNA was prepared with deproteinization, digestion and dialysis of nuclei and chromosomes. The results showed that concentration of the HMW DNA from 200 root tips (10 plugs) was about 4–20 ng/µL (4 ng/µL in chromosomes DNA and 20 ng/µL in nuclei DNA). The average insert fragment size was more than 100 kb after linage, transformation and insert fragment examination by pulse field gel electrophoresis for HMW DNA from nuclei. The similar results were obtained by HMW DNA prepared from chromosomes. It is confirmed that the method is applicable to prepare HMW DNA for construction of BAC library.

Key words: High molecular weight DNA, Sucrose gradient centrifugation, Synchronization of cell cycle, Chromosome flow sorting, BAC

CLC Number: 

  • S512
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