Transfer the product to a sterile microcentrifuge tube (50 ml) with 7 ml TE buffer.Īdd 700 μl 20% SDS and 800 μl of proteinase K (20 mg/ml), vortex for 1 min, then incubate at 55℃ for 60 min (the final concentration of proteinase K is 20 μg/ml). Put 1–2 g of wet cell mass into mortar, take a suitable amount of liquid nitrogen to cover the mass, and grind to freeze. This method is always used for mass extraction of DNA. Extraction of genomic DNA by grinding with liquid nitrogen Then dry at room temperature or at a higher temperature (≤55☌).Īdd 50 μl of 1×TE buffer to dilute the DNA (depend on the volume of DNA), preserve it at –20℃ for later use.Ģ.3.4. The aqueous phase containing the community DNA was transferred to another sterile microcentrifuge tube.Īdd 50 μl of 3 mol/L sodium acetate (pH 4.8–6.2), vortex gently, add 500 μl of isopropanol, or 800 μl ethylalcohol, then keep in the room temperature for more than 10 min or put it at 4℃ for 30 min to 2 h or overnight if necessary.Ĭentrifuge for 10 min (12,000 rpm), discard the supernatant, add 200 μl of 70% ethanol, shake slightly, then centrifuge for 5 min (12,000 rpm), discard the ethanol. Supernatants are transferred to fresh microcentrifuge tubes after centrifugation at 10,000 × g for 5 min at room temperature.Īdd 550 μl of mixer of phenol: chloroform: isoamyl alcohol (25:24:1), vortexing for 1 min, centrifuge for 10 min (12,000 rpm). This method is frequently used and suitable for most actinobacteria.ĥ0 mg of the pretreated cell samples are suspended in 480 μl TE buffer and 20 μl of lysozyme solution (50 mg/ml), then put them in shaker at 37℃ for overnight.Īdd 50 μl of 20% SDS and 5 μl of proteinase K (20 mg/ml), vortexing for 1 min, then put incubate at 55℃ for 60 min. In addition, avoiding contamination of exogenous DNA is also important. There are a lot of DNA enzymes in the environment that can digest the DNA or RNA, therefore some material used in the extraction has to be sterilized and enzyme inhibitors should be added in the extraction buffer at the same time. To ensure the quality of DNA, the following should be noticed: firstly, to avoid high temperature secondly, to control the pH at a certain pH range (pH 5–9) Thirdly, maintain the ionic strength of buffer which is of significance to maintain the space configurations of DNA And lastly, reduce the disruption of DNA in the course of extraction by physical factors, such as high speed oscillation, mixing and freezing–thawing. Otherwise, it is difficult to get the right result. Therefore, to ensure the quality of the DNA in the preparation of DNA samples is of great significance.
![change nuclotide contig in bioedit change nuclotide contig in bioedit](https://i.ytimg.com/vi/h6Ox9WOTBJ4/maxresdefault.jpg)
The principle of extraction and purification of genomic DNAĭNA contains all the genetic information which is all stored in the primary structure of DNA. However, genomic age put forward that some genomic characteristics have great potential in the taxonomy of bacteria and archaea as a substitute for the traditional method of determination of G+C content mol% and the labour-intensive DNA–DNA hybridization (DDH) technique. Some other molecular methods have been used in the classification of prokaryotes, such as multilocus sequencing typing (MLST), SDS-PAGE analysis of whole cell soluble proteins, secondary structure and signature nucleotides analysis of variable areas of the 16S rRNA gene. 16S rRNA gene was the best target molecule for studying the phylogenetic relationships because it is present in all the bacteria, functionally constant and composed of highly conserved as well as more variable regions. The advent of DNA amplification and sequencing techniques, in particular of the 16S rRNA gene, constituted the crucial criteria forward for determining the taxonomic status of prokaryotes, greatly increased the rate of discovering novel species and now routinely carried out as the first step in identifying novel organisms.
![change nuclotide contig in bioedit change nuclotide contig in bioedit](https://image.slidesharecdn.com/bioinfomaticslab-170715151606/95/bioinfomatics-laboratory-28-638.jpg)
Chemotaxonomy and DNA–DNA hybridization techniques were widely used subsequently. Later, physiological and biochemical properties of bacteria were also used for this purpose.
![change nuclotide contig in bioedit change nuclotide contig in bioedit](https://img.informer.com/screenshots/3524/3524380_2.jpg)
Initially, taxon of actinobacteria is based on phenotypic markers such as morphology, growth requirements or pathogenic potential.
![change nuclotide contig in bioedit change nuclotide contig in bioedit](https://bitesizebio.com/wp-content/uploads/2016/01/Figure-1.jpg)
Currently, the taxonomy and identification of prokaryotes rely on polyphasic combinations of phenotypic, chemotaxonomic and genotypic characteristics.