The high-quality genome DNA extraction of Proscopic SPP for complicated polymerase chain reaction amplification (PCR), because of the high percentage of secondary metabolites that bind or settle with nucleic acid. In this study, we report the modified sodium dodecyl sulfate / phenol sodium protocol that eliminates the use of liquid nitrogen in the process of maceration, β-mercaptoethanol in buffer extraction, and the steps of ethanol precipitation.
A₂₆₀ / a₂₈₀ absorbance ratio of isolated DNA is around 2.0 to 1.9, indicating that the DNA fraction is pure and can be used for further PCR analysis. DNA isolated by this quality protocol is sufficient for molecular applications; This technique can be applied to other organisms that have similar substances that inhibit DNA extraction. Finally, this proposal is an alternative method that is fast, inexpensive, and effective for genomic DNA isolation from Prosopist leaves SPP, even in low-tech laboratories.
Safer DNA extraction of plant tissue uses buffer sucrose and glass fiber filters.
For some plant species, DNA experiments and downstream experiments are hampered by various chemicals such as polysaccharides and polyphenols. This brief communication proposes a solvent extraction method (except for ethanol). This method consists of initial washing steps with Ste Buffer (sucrose 0.25 m, 0.03 m, 0.05 M EDTA), followed by DNA extraction using a glass fiber filter. The advantage of this method is safety and low costs.
The purity of the DNA solution obtained using this method is not necessarily as high as obtained using the STE / CTAB method, but sufficient for PCR experiments. These points are indicated empirically with two species, Japanese Speedwell and General Dandelion, which has been proven DNA is difficult to strengthen through PCR in previous research.
The optimization of high CTAB-throughput-based protocols for the extraction of QPCR class DNA from rumen fluid, plant and culture of bacteria.
The quality and results of DNA extracted are very important for most downstream applications in molecular biology. In addition, molecular techniques such as quantitative real-time PCR (QPCR) are increasingly widespread; Thus, the validation and comparison of cross-laboratory data requires standardization of upstream experimental procedures. The DNA extraction method depends on the type and size of the initial material used.
Thus, DNA extraction templates can be said to be the most significant variable when comparing data from different laboratories. Here, we explain the reliable, cheap, cheap and fast DNA purification method that equally applies to small or high DNA purification or high throughput. The protocol depends on CTAB-based buffers for cell lysis and further DNA purification with phenol: chloroform: isoamyl alcohol.
The protocol has been successfully used for DNA purification of rumen fluid and plant cells. In addition, after a few changes, the same protocol is used for large-scale DNA extraction from pure culture of gram-positive bacteria and gram negative. The DNA results obtained by this method exceeded which from the same sample using a commercial kit, and the quality was confirmed by a successful QPCR application.
Ancient DNA extraction from plants.
Various protocols for DNA extraction from archeological and paleobotanic plant specimens have been proposed. This is not surprising given the range of Taxa and the type of network that can be conserved and variations in conditions where the preservation can occur. Commercially available DNA extraction kits can be used to restore the DNA of ancient plants, but modifications to standard approaches are often needed to increase results.
In this chapter, I explained two protocols to extract DNA from a small amount of ancient plant tissue. The CTAB protocol, which I recommend used with single seeds, utilizes the incubation period in the next chloroform extraction buffer and extraction followed by DNA purification and suspension. PTB protocol, which I recommend used with pumpkin skin and similar networks, uses overnight incubation powder networks in extraction buffers, network removal by centrifugation, and DNA extraction from buffers using commercial DNA extraction kits.
The DNA genome extraction of the drug factory available in Malaysia uses an enhanced triomic extraction kit (TM).
DNA extraction was carried out on 32 samples of drug factories available in Malaysia using a triomic extraction kit (TM). The number is 0.1 g of flowers or young leaves with liquid nitrogen, ledsed at 65 ° C in the RY1 (plus) buffer and followed by RNASE care. Then, the RY2 buffer was added to the sample and completely mixed with Vortex before the removal of cell debris with centrifugation.
The supernatant was transferred to a fresh microcentrifuge tube and a 0.1 volume RY3 buffer added to each supernatant transferred. The mixture is applied to rotate the column followed by a centrifugation step to remove the buffer and other residues. Washing steps were carried out twice by applying 70% ethanol to the spin column. The DNA genome from the sample was restored by applying a 50 μL buffer to the membrane of each spin column, followed by a centrifugation step at room temperature.
Modification of Triomic Extraction Procedures (TM) is done by adding chloroform: isoamil alcohol (24: 1) steps in extraction procedures. DNA genoms extracted from most of 32 samples showed a total increase in results when chloroform: isoamyl alcohol (24: 1) steps applied in the extraction procedure trioMiko.
This preliminary study is very important for molecular medication plants available in Malaysia because DNA extraction can be completed in a shorter period of time (within 1 hour) compared to manual extraction, which requires applying phenol, chloroform, and ethanol precipitation, and requires. 1-2 days to finish.