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FAQ—Nucleic Acid Extraction, Purification and Detection
FAQ——Questions related to nucleic acid extraction, purification, and electrophoresis
| Q01: | What is the principle of DNA genome extraction? |
| A01: | After lysing cells or tissues, protease K and RNase are used to remove proteins and RNA, respectively. Subsequently, DNA is precipitated with ethanol. Following this, a silica-based material adsorption column is employed to specifically adsorb the DNA. The principle of silica-based adsorption column adsorption involves binding at high salt and low pH conditions, followed by elution at low salt and high pH conditions. |
| Q02: | What is the reason for the low concentration of genomes I extracted with the genome extraction kit? |
| A02: | The sample used for extraction is preferably fresh, and the extraction effect of fresh samples is better. Extend proteinase K and RNA enzyme treatment time to ensure cleavage efficacy. The column adsorption time was extended to allow sufficient adsorption of DNA onto the silicon matrix adsorption column.The Eluent needs to be preheated in advance, and the volume of eluent can not be too small, otherwise the elution is not sufficient, which will also lead to low concentration. |
| Q03: | Why is the plant genome extraction kit not effective? |
| A03: | Our plant genome extraction kit is not suitable for the genome extraction of polysaccharide polyphenols samples, so you can check your plant samples if it contain polysaccharide polyphenols. |
| Q04: | How to use DNA extraction reagents in P1012, P1013 and P1021 ? |
| A04: | These?products?are?all?used?for?DNA?extraction?and?containthe?same?components,?but?in?different?proportions.?Choose?according?to?your?specific?needs.?When?using?them,?simply?pipette?the?lower?layer?of?the?liquid. |
| Q05: | What are the differences between T0250 DNA extraction phenol reagent and W0250 RNA extraction phenol reagent, and how are they used? |
| A05: | The DNA extraction phenol reagent has a pH greater than 7.8 and is primarily used for DNA separation, typically in combination with chloroform and isoamyl alcohol to achieve DNA purification. The RNA extraction phenol reagent has a pH less than 5.5 and is mainly used for RNA separation, also requiring the use of chloroform and isoamyl alcohol to separate RNA. When using these products, always pipette the lower layer of the solution. If the solution turns red or brown, it indicates oxidation and the product should no longer be used. |
| Q06: | What are the purposes and differences between TE Buffer and TAE Buffer? |
| A06: | The primary components of TE Buffer are Tris and EDTA, which are commonly used for dissolving or preserving DNA. On the other hand, TAE is the most widely employed buffer system and serves primarily as the electrophoresis buffer for agarose gel electrophoresis experiments. |
| Q07: | What product can be used to dissolve RNA? |
| A07: | R1600 Enzyme-Free Sterile Water is water treated with DEPC (diethyl pyrocarbonate) and subsequently subjected to high temperature and high pressure, during which the DEPC in the water decomposes. It can be used for dissolving RNA or in molecular biology experiments sensitive to nucleases, such as cDNA synthesis and in vitro transcription. SR0080 RNAsaver is a reagent that can be directly used to dissolve RNA precipitates. The dissolved RNA can undergo normal electrophoresis. However, RNAsaver inhibits subsequent enzymatic reactions. Therefore, if the RNA is intended for reactions such as RT-PCR, it is necessary to pre-precipitate and remove RNAsaver using LiCl (by adding 1/3 volume of LiCl to the solution, centrifuging at 12000-15000 g for 20-30 minutes at 4°C, discarding the supernatant, and washing the RNA precipitate once with 75% ethanol). |
| Q08: | What are the reagents related to RNA extraction? |
| A08: | R1100 Trlquick Reagent is a total RNA extraction reagent used for lysing tissue or cellular samples to extract total RNA. It requires the combination with chloroform and other related reagents to co-extract and purify RNA precipitates. R1200 Total RNA Extraction Kit contains lysis buffer and adsorption columns, while reagents such as chloroform and absolute ethanol need to be prepared separately. Chloroform can be replaced by P1014 Nucleic Acid Extraction Reagent at a ratio of 24:1. R2000 RNA Virus Genome Extraction Kit is primarily used for extracting RNA virus genomes from serum, cell supernatants, and lymph fluid. It is not suitable for extracting RNA virus genomes from tissues or cells. SR0020 RNAwait (Non-Freezing Tissue RNA Preservation Solution) is used to preserve RNA in tissues or cells, preventing degradation by RNases. SR0040 Solid-Phase RNase Remover can be applied to eliminate RNases on the surfaces of experimental utensils, instruments, and other laboratory items, safeguarding RNA from degradation during experiments. SR0060 Liquid-Phase RNase Remover contains special compounds that inactivate trace amounts of RNases potentially contaminating solutions, making it suitable for dissolving RNA precipitates. It can also be used to treat amino-containing solutions that cannot be treated with DEPC, such as Tris and MOPS. SR0080 RNAsaver Long-Term RNA Preservation Solution can be directly used to dissolve RNA precipitates. The dissolved RNA can undergo normal electrophoresis, but RNAsaver will inhibit subsequent enzymatic reactions. R1600 Enzyme-Free Sterile Water is water treated with DEPC and subjected to high-temperature and high-pressure sterilization. The DEPC in the water has been decomposed, making it suitable for dissolving RNA or for molecular biology experiments sensitive to nucleases, such as cDNA synthesis and in vitro transcription. D8210 RNase Remover is a chemical modifier of RNases that inhibits enzyme activity by reacting with the imidazole ring of the active group histidine in RNases. |
| Q09: | Can DNA virus genome extraction kits and RNA virus genome extraction kits be used to extract viral genomes from cells or tissues? |
| A09: | No, our company's viral genome extraction kits are primarily designed for extracting RNA virus genomes from serum, cell supernatants, and lymph, and are not suitable for extracting RNA virus genomes from tissues such as cells. |
| Q10: | What are the main differences between the D1100 Mini Plasmid Extraction Kit and the D1110 Maxi Plasmid Extraction Kit? |
| A10: | The extraction principles of the two kits are the same. The main difference lies in the amount of plasmid that can be extracted in each operation. The Maxi Plasmid Extraction Kit (D1110) allows for a larger amount of plasmid to be extracted per run compared to the Mini Plasmid Extraction Kit (D1100). |
| Q11: | Can the D1600 Bacterial Genomic DNA Extraction Kit be used to extract DNA from Gram-positive bacteria? |
| A11: | The D1600 is primarily designed for extracting genomic DNA from Gram-negative bacteria. Since Gram-positive bacteria have thicker cell walls, if the intention is to extract genomic DNA from Gram-positive bacteria, the customer would need to purchase lysozyme separately to lyse the cell walls prior to performing the extraction process. |
| Q12: | What is the primary purpose of the N1030 Centrifugal Filter Column? |
| A12: | The N1030 is primarily used in gel recovery experiments for filtering out gel fragments. |
| Q13: | What are the main functions of each component in the D1100 Mini Plasmid Extraction Kit? |
| A13: | RNase A primarily serves to remove RNA. Solution I is mainly used to resuspend bacterial cells, facilitating the subsequent lysis of bacterial cells. Solution II is primarily responsible for lysing bacterial cells, releasing the genomic DNA and plasmids. This step should be performed gently and slowly to prevent genomic DNA fragmentation and contamination of the plasmids. Solution III primarily adjusts the pH, precipitates proteins and genomic DNA, providing a high-salt, low-pH environment for the subsequent adsorption of plasmids. The wash buffer's primary function is to remove impurities from the plasmids. The elution buffer is used to elute the plasmids from the adsorption column. The adsorption column is mainly used for the purification of plasmids, adsorbing the plasmids. |
| Q14: | What are the main functions of each component in the D1600 Bacterial Genomic DNA Extraction Kit? |
| A14: | RNase A primarily serves to degrade RNA. Protease K degrades proteins, separating them from the genomic DNA. Solution A is used to resuspend and lyse bacterial cells, releasing the genomic DNA. Solution B primarily adjusts the pH, providing a high-salt, low-pH environment for the subsequent adsorption of the genomic DNA. The wash buffer's main function is to remove impurities from the genomic DNA. The elution buffer is primarily used to elute the plasmids (note: typically, the elution buffer in a bacterial genomic DNA extraction kit would be used to elute the genomic DNA, not plasmids, as stated in the original answer. This may be a misstatement in the context of this kit). The adsorption column is primarily used for the purification of the genomic DNA, adsorbing the genomic DNA. |
| Q15: | What are the main categories of nucleic acid electrophoresis and their respective application directions? |
| A15: | Nucleic acid electrophoresis is primarily divided into agarose gel electrophoresis and polyacrylamide gel electrophoresis. Agarose Gel Electrophoresis: This method utilizes agarose as the supporting medium for gel preparation. Within a certain range, the general principle is to use low-concentration agarose gels for separating larger DNA fragments and high-concentration agarose gels for separating smaller fragments. Separation range: 0.2-50 Kb. Polyacrylamide Gel Electrophoresis: Polyacrylamide gel electrophoresis achieves separation based on differences in the charge, molecular size, and shape of the electrophoresis samples. It combines molecular sieve and electrostatic effects, resulting in higher resolution than agarose gel electrophoresis. It can separate DNA fragments that differ by as little as one nucleotide. (1) Separation range: 1-1000 bp (2) Polyacrylamide gel electrophoresis is primarily used for nucleic acid precast gels, which are further classified into denaturing and non-denaturing. Denaturing: Used for separating single-stranded DNA fragments Non-denaturing: Used for separating double-stranded DNA fragments. |
| Q16: | Can low-melting-point agarose be used for cell culture? |
| A16: | Our low-melting-point agarose is primarily intended for gel recovery experiments, and we have not used it for cell culture. |
| Q17: | What are the differences between TAE, TBE, and MOPS buffers? |
| A17: | 1, TAE: It has a small buffer capacity but excellent solubility, allowing for the preparation of high-concentration stock solutions, which are convenient to use and easy to store. 2, TBE: It has a large buffer capacity, allowing for fewer buffer changes over time. However, its solubility is not as good, and it is typically only prepared as a 5×stock solution, which is not suitable for long-term storage due to the risk of precipitation. It is preferred for long-duration electrophoresis and the separation of small DNA fragments (less than 1 kb).TAE buffer system is also recommended for electrophoresis when recovering DNA fragments. 3, MOPS (3-(N-morpholino)propanesulfonic acid): It is a biological buffer used to prepare RNA electrophoresis buffers. |
| Q18: | What is the working concentration of TBE buffer? |
| A18: | The working concentration of TAE buffer and MOPS buffer is 1×, while the working concentration of TBE buffer is 0.5×. |
| Q19: | What is the difference between D1010 6×DNA Loading Buffer and D1020 10×DNA Loading Buffer? |
| A19: | Both are DNA electrophoresis loading buffers, but they differ in their concentration factors. When using them, they need to be mixed with the sample to achieve a final concentration of 1× for loading. |
| Q20: | What causes the bands to curve after running electrophoresis with G8142 Nucleic Acid Dye? |
| A20: | The G8142 Nucleic Acid Dye has high sensitivity. Generally, commercially available markers perform better when diluted 5-10 times before electrophoresis. For samples with high concentrations, appropriate dilution is also necessary before electrophoresis to achieve better results. |
| Q21: | What are the nucleic acid dyes commonly used in nucleic acid electrophoresis? |
| A21: |  |
| Q22: | What are the differences between the various staining methods used for nucleic acid dyes |
| A22: |  |
| Q23: | Why can I see two indicator bands after running electrophoresis with DNA loading buffer? |
| A23: | Our company's DNA loading buffer contains two indicator dyes: xylene cyanol and bromophenol blue. The leading dye is bromophenol blue, which serves as the primary indicator. |
| Q24: | What reagents are required to observe the quality of RNA extraction during RNA electrophoresis? |
| A24: | If the sole purpose is to observe the quality of RNA extraction, the following reagents can be used for agarose gel electrophoresis: A8201 agarose, T1060 50× TAE buffer, DNA marker, and either DNA or RNA loading buffer. |
| Q25: | What is the difference between R1055 2×RNA Loading Buffer (denaturing) and R1050 5×RNA Loading Buffer (non-denaturing)? |
| A25: | The denaturing loading buffer contains denaturants that open the secondary structure of RNA, while the non-denaturing loading buffer does not contain denaturants and thus preserves the secondary structure of RNA. |
| Q26: | What is the difference between non-denaturing and denaturing precast gels for nucleic acid electrophoresis? |
| A26: | Both non-denaturing and denaturing precast gels are polyacrylamide gel electrophoresis systems. Denaturing precast gels contain urea as a denaturant, which converts nucleic acids into single strands, enabling the separation of single-stranded nucleic acids. Non-denaturing precast gels, on the other hand, do not contain denaturants and preserve the double-stranded or secondary structure of nucleic acids, allowing for the separation of double-stranded nucleic acids or those with secondary structures. |
| Q27: | What are the dimensions of precast nucleic acid gels and what electrophoresis chambers can they be used in? |
| A27: | The dimensions of precast nucleic acid gels are listed on our official website. The gels come in 10-well and 15-well formats with identical dimensions. Customers can compare the size of their gel casting plates with ours to determine compatibility with their electrophoresis chambers. |
| Q28: | Can R8021 RNase A (Ribonuclease A) and R8020 RNase A (Imported) be directly dissolved in water? |
| A28: | No, RNase A is insoluble in neutral water. It must be dissolved according to the instructions provided, which often involve boiling to activate the enzyme. Therefore, it is crucial to follow the prescribed preparation method. |
| Q29: | Are R8021 RNase A and R8020 RNase A (Imported) free of DNase enzymes? |
| A29: | Both R8021 and R8020 Ribonuclease A powders are not DNase-free, however, during the preparation process, ribonuclease A (RNase A) requires boiling, which inactivates any DNase present. Therefore, the prepared RNase A solution is devoid of DNase. R1030 RNase A Solution (10mg/ml) is DNase-free. |
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