Rapid Crude Extraction of Genomic DNA from Solanum lycopersicum for PCR

doi:10.13560/j.cnki.biotech.bull.1985.2021-1123

Abstract

Abstract:

It is necessary to extract plant genomic DNA and perform PCR cloning when conducting the studies of molecular biology on plants. Currently,the CTAB（Cetyltrimethyl Ammonium Bromide）is commonly used to extract plant DNA in laboratories,but the steps are relatively cumbersome and require multiple organic solvents. Specifically,it takes a long time when there are many samples. The DNAs from multi-species were extracted by NaOH,and this study introduces a method of extracting plant genome DNA,which is faster,economical,safer and more efficient. The major steps include the followings：Quickly grinding the plant tissues using liquid nitrogen;quickly extracting DNA by alkaline lysis（0.5 mol/L NaOH）;neutralizing the extracted DNA sample with TE buffer;and performing PCR with neutralized DNA as template. Using this simple DNA extraction method and combining PCR detection,the rapid preliminary identification of transgenic S. lycopersicum was completed. Furthermore,it was confirmed that this NaOH method could be used for extracting DNA from the different tissues of S. lycopersicum. The entire extraction process was simple and of short-time. Thereby,DNA extraction for a large number of plant samples could be completed in a short time without using toxic regents such as chloroform and isopropanol etc. The completion time of DNA extraction is about 1/5 of the time while using CTAB method. In addition,the extracted DNA from different tissues are of good integrity and high quality,which meet regular PCR detection,which can be used for gene cloning and for transgenic tomato screening. This method demonstrates potential utilization value and prospects.

Key words: Solanum lycopersicum, NaOH, DNA extraction, PCR, identification of transformant

SHEN Heng, LIU Si-hui, LI yue, LI Jing-tao, LIANG Wen-xing. Rapid Crude Extraction of Genomic DNA from Solanum lycopersicum for PCR[J]. Biotechnology Bulletin, 2022, 38(6): 74-80.

Figures/Tables 9

Table 1 Comparison of NaOH and CTAB method for extracting S. lycopersicum DNA

DNA提取方法 Methods for DNA extraction	仪器 Instrument	危险试剂 Hazardous reagent	累计时长Total time/h
NaOH	组织研磨仪	NaOH	约0.5
CTAB	组织研磨仪,水浴锅	CTAB,氯仿,异戊醇,乙醇	约2.5

Fig. 1 Quantitative detection of extracted genomic DNA from S. lycopersicum with different NaOH concen-trations

Fig. 2 PCR detection of S. lycopersicum genome with diff-erent NaOH treatment time

Fig. 3 Quantitative detection of extracted genomic DNA from S. lycopersicum with different methods

Fig. 4 PCR detection of extracted genome by different methods

Fig. 5 PCR detection of the extracted leaf genome of S. lycopersicum transformants using NaOH method

Fig. 6 PCR detection and sequencing verification of S. lycopersicum transformants A：PCR detection of the extracted genome of S. lycopersicum transformants using NaOH and CTAB methods. B：Sequencing analysis of PCR products from S. lycopersicum transformant 1

Fig. 7 DNA extraction and PCR detection from the differ-ent tissues of S. lycopersicum seedlings A：Different S. lycopersicum tissues. B：PCR results of DNA from different tissue samples. The detected gene is SlActin

Fig. 8 Schematic diagram illustrating the process of DNA extraction and PCR detection from S. lycopersicum tissues

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