Soil DNA Extraction Part II: Chemistry
Then the previous article on how to improve the soil DNA extraction rate topic. We have thoroughly talked about the importance of sample grinding and cracking, the choice of grinding bead type, grinding equipment, and lysis buffer. From this you can also find that the cracking step is the most worthwhile place to increase the yield. After the grinding and lysis, the final DNA is extracted in advance, and it needs to be cleaned and removed. This step is mainly done by the inhibitor removal factor (IRT). Following the flow of the instructions, after the removal of the inhibitor is completed, the next paragraph of the article is to extract the DNA using a chemical solution and a silica spin column. Remove the inhibitor: After the soil sample is ground and broken, the PCR inhibitor removal can be performed. The so-called humic acid is actually a substance that makes the sample brown. If there are plant residues or even biofilms in the sample, the impurities will also contain polysaccharides. MO BIO and Powersoil kits excel in their peers and similar kits because of their inhibitory factor removal. MO BIO Labs has developed a patented technology called Inhibitor Removal Technology (IRT). It can precipitate humic acid, polysaccharides, polyphenols, etc. from cell lysates. The IRT technique is a two-step process that removes proteins and other residues and then removes insoluble macromolecular flocculents. After the IRT treatment, the sample looks much clearer. PowerSoil and PowerWater have been experimentally calculated to use a sufficient amount of IRS to deal with the most difficult problem soil. If an inhibitory factor (PCR amplification assay) is still present, repeat the IRS step. The first step of IRT requires low temperatures to enhance flocculation. In the second step, we recommend not extending the incubation time beyond 5 minutes. Tests have shown that too long incubation times reduce DNA yield. midpoint? If you want to temporarily suspend the experiment, the time point is best after the end of the IRS, before adding the binding solution. Store the lysate at -20 ° C until the next day and then do the silica gel spin column binding. Bind to the silicone film: At this point, the DNA can already be extracted through a silica gel membrane. Generally, the cell lysate obtained in the previous step should be clear (if the soil has a lot of organic matter, it may be slightly yellowish). In order to adsorb DNA onto the silica gel film, the help of chaotropic salts is required. The ratio of binding solution (C4 solution) to dissolved product is another key point in yield. Too much binding fluid will result in excessive recovery of degraded RNA; if too little, the recovery of large molecular weight genomic DNA will be low. Therefore, we recommend a 2ml Tube, put 750μl of lysate, 1.2ml of binding solution. If more than 750 μl of lysate is found during the experiment, you will need to increase the amount of binding solution accordingly. Or the binding solution (C4 solution) is twice the volume of the dissolved product, and this ratio is most suitable. In this case, separate the lysate into two 2ml Tube collection tubes, or use a large Tube (5mL or 15mL) and ensure that the solution is fully mixed. Vacuum pump adapter (Vacuum Manifold), optional: Normally, the adsorption binding to the spin column requires 3 additions. One way to increase productivity is to try the PowerVac Manifold System. If you have a vacuum manifold in your lab, you only need one PowerVac Mini Spin Filter adapter. We use this method to improve the speed of work in the laboratory. If you need to handle more than the recommended amount of 750 μl of lysate and the corresponding increase in binding solution, then using the vacuum manifold can save a lot of time while loading 4-5 times. Cleaning: Because of IRT, all contaminants in the soil have been removed without the need for additional high salt like other branded kits. The main purpose of this step is to remove the chaotropic salts remaining on the column. If these chaotropic salts are not removed, the DNA will not elute very easily, and even if it is eluted, it will be contaminated by sputum. The cleaning buffer contains an alcohol component that dissolves and washes away residual salts. It is usually done once. If the 260/230 reading is found to be low (230 absorbance is high in the Nanodrop reading), please clean it again. If the wash buffer is found to be used up, 100% absolute ethanol can also be used as an alternative to rinsing the filter. 100% absolute ethanol is used in the vacuum manifold operating instructions. After rinsing, remember to dry the alcohol on the spin column. Because residual alcohol can affect the extraction efficiency of DNA. Extraction: The final step was to release the DNA into 10 mM Tris pH 8.0 buffer. DNA is easily degraded in a neutral or weakly alkaline environment. You may consider using water to dilute it, but usually the water pH is low (about 4-5) and the effect may not be ideal. A small reminder of the increase in yield during the extraction process is to incubate the spin column filter at room temperature before centrifugation after adding the buffer. Incubation for 1-5 min can help resuspend the concentrated DNA to a smaller volume. However, it is recommended that the final volume of DNA extraction should not be less than 50μl, otherwise a large amount of DNA will remain. Now, your DNA is ready to be PCR amplified or run. common problem: How much DNA is usually in the soil? After discussing so much, what you may want to ask most is, how much DNA does the soil generally contain? The answer is uncertain. The water content of the soil, the amount of organic matter, and the location of the collection can all be affected. In our laboratory, "general soil" such as garden soil, DNA yield can reach 2-5μg / 0.25g (each sample). We also calculated the field soils such as strawberry gardens, the yield is very low, know 0.25μg / 0.25g (each sample). For soil types with low levels of organic matter such as sand and clay, the yield may be lower. What can I do to increase the yield of clay and sand? One theory of clay and sand suggests that some of the released nucleic acids are firmly bound to the soil itself. At the end of the article, several pre-treatment methods for preventing microbial DNA loss are listed. This includes the use of skim milk (1). There is evidence that divalent cations play a key role in DNA adsorption to the soil surface (2). Therefore, some customers added EDTA to the grinding tube during the lysis step and successfully obtained a final concentration of 50 mM DNA. to sum up: The article contains all the technical tips and techniques we know about extracting soil DNA. In general, different soil types have their characteristics and microbial content to predict DNA yield. The key step in achieving high yields and complete DNA is the grinding and adsorption binding phase. If your final yield does not meet expectations, the solution will focus on two points. Please avoid blindly increasing the initial amount of soil samples and not getting more DNA. We are always happy to hear your opinions and opinions on using the MO BIO kit for better results. Please write to us and tell us your unique skills in extracting DNA from soil samples. Thank you for your patience to read! references: COVID-19 Antigen Test Cassette (Nasal Swab) This detection kit applies immunoassay to directly detect the presence of virus and is possible to reach large-scale testing of up to millions of tests one day COVID-19 Antigen Test Cassette (Nasal Swab) NINGBO AUTRENDS INTERNATIONAL TRADE CO., LTD , https://www.metests.com Author: Suzanne kennedy Source: US MoBio Laboratory [Font Size: medium and small]