DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid Nitrogen and Phenol.

DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid Nitrogen and Phenol.

Mangrove and Salt Marsh species are known to synthesize the width spectrum of polysaccharides and polyphenols including flavonoids and other secondary metabolites that disrupt the extraction of pure genome DNA. Despite a number of plant insulation protocols there are plants, extract DNA from mangrove and swamp salt species is a challenging task.

This study illustrates the CETYL Trimethylammonium Bromide (CTAB) protocol specifically and is specifically extracted to extract DNA from plants rich in polysaccharides and secondary metabolites, and the protocol also does not include the use of liquid nitrogen and expensive toxic phenol phenol. DNA purity extracted is very good as evidenced by the A260 / A280 ratio from 1.78 to 1.84 and the ratio of A260 / A230> 2, which also suggests that preparation is quite free of protein and polyphenolics / polysaccharide compounds.

The concentration of DNA ranges from 8.8 to 9 μg μl (-1). The extracted DNA is receiving RAPD, digestive restrictions, and amplification of PCR gene barcodes (MATK and RBCL). Optimized methods are suitable for dry and fresh leaves. The success of this method in obtaining a high-quality genome DNA shows the application of this extensive method.

Simple method for normalization of DNA extraction to increase quantitative detection of Oomycete Pathogen of soil plants by real-time PCR.

Most of the current research into the quantification of oomycet pathogens that are transmitted through land does not have determination of DNA extraction efficiency, it may lead to the estimated DNA quantity is wrong. In this study, we developed a convenient method using the artificially synthesized DNA DNA sequence originating from the mitochondrion gene NADH Dehydrogenase Subunit 2 Thunnus Thynnus as a control to determine the efficiency of DNA extraction.

DNA controls added to the ground and then extracted along with the DNA of the Genom of the Soil. DNA extraction efficiency is determined by DNA control. Two different DNA extraction methods are compared and evaluated using various types of soil, and the commercial kit is proven to provide more consistent results. We use DNA controls combined with real-time PCR to measure Oomycete DNA of 12 full natural land.

Target DNA concentrations that can be detected three to five times higher after normalization. Our test also shows that extraction efficiency varies based on sample-to-sample and <50%. Therefore, the method introduced here is simple and useful for accurate quantification of the pathogenous oomycetes that are transmitted through the ground.

Oomycetes includes many important plant pathogens. The accurate quantification of these pathogens is very important in managing diseases. This research reported an easy method of utilizing external DNA controls for Normalization of DNA extraction by real-time PCRs. By combining two efficient land DNA extraction methods, the quantification method developed dramatically increases results. This research also proves that the method of normalization developed is needed and useful for accurate quantification of oomycete land plant pathogens.

Extraction of plant DNA and metagenomics from mukilaginous seeds.

Pulp that surrounds the seeds of some rich fruit will be mucus, carbohydrates, etc. Some seeds are rich in protein and polyphenol. Fruit seeds, such as cocoa (theobroma cocoa) and cupowsu (theobroma grandiflorum), experience fermentation to develop taste. During fermentation, ethanol is produced [2-6]. All of these compounds are considered as disturbing substances that inhibit DNA extraction [4-8].

The protocol commonly used in DNA extraction in the origin of the plant origin is used, but without results. Thus, the protocol for DNA samples in different conditions that can be used for similar samples is developed and applied with success. The protocol was originally explained for RNA samples by Zeng et al. [9] And with the changes proposed by Provost et al. [5] Adapted to extract DNA samples from described.

DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Polysaccharides without Using Liquid Nitrogen and Phenol.

However, some modifications have been proposed: • The sample was originally washed with ether petroleum for the removal of the fat phase. • RNASE is added to the extraction buffer, while spermidine is removed. • Additional steps of extraction with NaCl 5 M, saturated NaCl and CTAB (10%) are included and rainfall is done with isopropanol, followed by washing with ethanol.

High quality Genomic DNA extraction protocol.

High quality genomic DNA isolation (GDNA) is an important technique in plant molecular biology. GDNA quality determines the reliability of the real-time polymerase chain reaction analysis (PCR). In this paper, we report high-quality GDNA extraction protocols that are optimized for real-time PCRs in various plant species. Done in the 96-sound block, our protocol provides a high throughput. Without the need for phenol-chloroform and liquid nitrogen or dry ice, our protocol is safer and more cost effective than traditional DNA extraction methods.

This method takes 10 mg leaf tissue to produce 5-10 μg of high quality GDNA. Spectral and electrophoresis measurements are used to indicate GDNA purity. DNA extracted qualifies in the digestive test of conventional restrictions and PCR enzymes. Real-time PCR amplification is quite sensitive to detecting GDNA at very low concentrations (3 pg / μL).

The standard curve of the GDNA dilution from the phenol-chloroform protocol we showed a better linearity (R (2) = 0.9967) compared to the phenol-chloroform protocol (R (2) = 0.9876). The results show that GDNA is of high quality and suitable for real-time PCR.

This secure GDNA GDNA extraction protocol can be used to isolate high-quality GDNA for real-time PCR and other downstream molecular applications.