1. The sample lysis effect is not good. Can increase the amount of lysis solution resulting better results?
For some samples, increasing the amount of lysis buffer used can indeed improve the lysis effect. However, in the process of nucleic acid extraction by magnetic bead method, as the volume of lysate increases, the probability of collision between magnetic beads and nucleic acid decreases, and the efficiency of nucleic acid adsorption by magnetic beads decreases, which may lead to problems such as the reduction of nucleic acid extraction amount and the reduction of nucleic acid extraction purity. The negative effects of increasing the amount of lysate used tend to outweigh the positives. If the sample lysis effect is not good, it can be explored by adjusting the lysis solution formula, method, time or reducing the sample amount.
2.How to avoid nucleic acid degradation during nucleic acid extraction?
The first thing to do is to avoid repeated freezing and thawing of samples. During nucleic acid extraction, choose enzyme-free consumables. At the same time, control the nucleic acid degradation caused by shear force, uneven surface of magnetic beads, high elution temperature and long extraction process during extraction process.
3. Can increase the amount of sample extracted to enhance the extraction effect?
In the case of the nucleic acid content of the sample is high and the lysis solution can be fully lysed, increasing the sample volume can increase the extraction effect. However, for samples with low nucleic acid content, increasing the sample volume will introduce more impurities and reduce the quality of the final nucleic acid extraction. For such samples, nucleic acid extraction can be performed after preliminary processing (such as concentration) of the sample.
4. Can increase the number of magnetic beads improve the yield of nucleic acid extraction?
When the amount of nucleic acid extraction is low, many researchers like to increase the amount of magnetic beads in the extraction process, thinking that this can adsorb more nucleic acids, thereby increasing the amount of nucleic acid extraction, but this method is often difficult to achieve the ultimate goal. If the amount of magnetic beads is too much, it will not be able to be uniformly dispersed into the solution system of each step, reducing its dispersibility, and leading to incomplete adsorption, washing and elution processes. Excessive magnetic beads will also adsorb more impurities, and there are no nucleic acid binding sites on the surface of the magnetic beads, which will provide more opportunities for impurities to bind. In general, the amount of magnetic beads given in the magnetic bead method nucleic acid extraction kit is enough to meet the common application scenarios. If the problem of low nucleic acid extraction yield happens, try to consider the cleavage binding, cleaning and elution processes.
5. The purity of nucleic acid extraction is low, and there are protein or salt residues. Can washing multiple times increase the result?
For this problem, the number of washings can be appropriately increased by one or two times. Excessive washing increases the amount of nucleic acid loss during washing. Under normal circumstances, the number of cleanings is 2 to 4 times the best.
6. Why do the magnetic beads that worked well, but perform badly after changing a kit?
Magnetic beads correspond to different extraction systems (such as extraction buffer and extraction process), and they do have different effects. The magnetic beads with excellent performance in the original system are the best combination after a series of screening and method exploration. If the magnetic beads are replaced with a new system, it is normal for the extraction effect to decrease. The extraction effect can be optimized by adjusting the Buffer composition and extraction process. Nanotein can provide technical services for matching magnetic beads with specific nucleic acid extraction kits, or screening the most suitable matching Buffer, to save R&D costs for teachers and speed up project progress.
7. How to improve the purity of nucleic acid extraction and prevent impurities from inhibiting downstream applications?
The purity of nucleic acid extraction is not high, and the main cause of contamination is protein and (or) salt residues. During the extraction process, proteinase K can be added to degrade the protein in the sample to reduce the residual contamination of the protein. At the same time, the nucleic acid is washed with a high concentration of organic solvent to reduce the inhibition of the enzyme activity by the salt residue.
8. How to improve nucleic acid extraction yield?
During the lysis process, the sample should be fully lysed to fully expose the nucleic acid. The amount of buffer in the system should be appropriate so that the magnetic beads and nucleic acids have sufficient contact opportunities. The number of cleaning processes should be controlled, and nucleic acid should not be lost due to excessive cleaning.
