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FAQ—Solarbio Plasmid Extraction
FAQ Documentation—Plasmid Extraction
| Q01: | Why are the bands not bright when staining with G8140 Green Fluorescent Nucleic Acid Dye (10000×)? |
| A01: | The Green Fluorescent Nucleic Acid Dye is particularly suitable for detecting large DNA fragments (greater than 1kb, with detection sensitivity comparable to EB). When the DNA fragments are smaller than 1kb, especially below 500bp, the detection sensitivity is lower than EB, and the dye may exhibit weak brightness or undetectable signals. For detecting small DNA fragments, please use G8142, which is suitable for all fragment sizes. Generally, add 10μL of dye per 100mL of gel. If the fluorescence appears weak, the amount of dye can be increased appropriately. |
| Q02: | When using G8142 GoldView II Nucleic Acid Dye for 1% agarose gel electrophoresis and gel recovery of PCR products, the electrophoresis results show that both the MARK and target bands are unclear and diffuse, making subsequent experiments impossible. |
| A02: | When using, please dilute the commercial Marker 5-10 times with 1×DNA Loading Buffer and load 1-10μl for better results (typically 5μl after 10-fold dilution). For samples to be detected, usually only 1-2μl is required. If the concentration is high, it must also be diluted accordingly, as high concentrations can cause uneven bands and affect migration rates. For gel recovery, the spot staining method is recommended. |
| Q03: | The extraction yield is insufficient. |
| A03: | Generally, increasing the sample volume is the simplest method. Additionally, the main principle of the adsorption membrane is low-temperature binding and high-temperature elution. Therefore, when the adsorption column is at rest, placing it at 4°C for 3-5 minutes before re-adsorption can enhance the extraction yield by increasing the number of binding cycles. If the extracted plasmid is small or has a low copy number, a small amount of 1/5-1/3 isopropanol can be added during the adsorption step to improve recovery (generally not recommended). |
| Q04: | What is the extraction yield of D1140? |
| A04: | Due to variations in plasmid copy numbers, the yield may not exceed 100. |
| Q05: | Are there any columns specifically designed for plasmid extraction? |
| A05: | Both N1010 and N1011-A can be used. |
| Q06: | Can N1011-B be used for plasmid extraction? |
| A06: | No. It is only suitable for genomic DNA extraction and can lead to falsely elevated plasmid concentrations when used for plasmid extraction. |
| Q07: | How much solution volume can the N1010 series columns accommodate? |
| A07: | Approximately 650-700 μl. |
| Q08: | Can your elution buffer be used for sequencing extracted plasmids? |
| A08: | Yes, generally, it has minimal impact. If unsure, sterile ddH2O can be used for elution instead. |
| Q09: | After extracting plasmids, transformation fails. |
| A09: | Generally, the first suspicion should be the competence of the cells. If the competence is confirmed to be okay, check if the wrong antibiotic was used or if there were issues during the operation. If the problem is confirmed to be with the plasmid, increase the amount of plasmid used for transformation (typically, only 1μl of plasmid at a concentration of around 500ng/μl is needed; if the concentration is low (<100ng/μl), the amount can be increased to 10μl). |
| Q10: | No bands are visible in PCR using extracted plasmids. |
| A10: | 1, Check if the primers are suitable. 2,Verify the suitability of the PCR program. 3, Examine if there were any issues during the operation. 4, Ensure the plasmid concentration is not too high (typically, 50-100ng/μl for PCR). |
| Q11: | Can antibiotics be added during plasmid transformation? |
| A11: | Yes, it won't affect the efficiency of plasmid transformation. |
| Q12: | No plasmid was extracted. |
| A12: | 1,The bacterial strain may be aging (try re-transformation or revitalizing the strain). 2,Insufficient lysis. 3,The bacterial strain may genuinely lack plasmids. 4,Check if there are precipitates in the reagents of the kit. 5,When adding elution buffer, ensure it is added directly onto the adsorption membrane and in sufficient volume. |
| Q13: | If there is an issue with either PCR or restriction enzyme digestion identification of the extracted plasmid, can it still be used? |
| A13: | No, it will not work. |
| Q14: | The size of the plasmid detected by gel electrophoresis using P3110 pET-28a(+) does not match the size specified in the manual. |
| A14: | To accurately determine the size of the plasmid by gel electrophoresis, it must first be linearized through single-enzyme digestion. Supercoiled or open-circular plasmids cannot accurately indicate the plasmid size. If the customer does not accept the single-enzyme digestion results, direct sequencing can be performed for detection. |
| Q15: | After using R1030 RNase A solution (10mg/ml) to extract plasmids from E. coli, RNA was not removed from the plasmid solution. |
| A15: | The working concentration of RNase A solution is 200μg/ml, while the customer's concentration is 10μg/ml. It is recommended to increase the usage amount. |
| Q16: | The plasmid is completely resistant to endonuclease cleavage. |
| A16: | 1,The enzyme activity may be low; increase the incubation time or enzyme quantity. 2,The plasmid and enzyme activity may not be compatible; optimize the digestion conditions. 3,The plasmid may be impure, containing inhibitors such as SDS, phenol, or EDTA. 4,The restriction sites on the plasmid may be mutated. 5,The DNA may not contain the recognition sequence for the enzyme. 6,The buffer may not be compatible with the enzyme. |
| Q17: | The plasmid is not completely digested by the endonuclease. |
| A17: | 1,The enzyme activity may be low; increase the incubation time or enzyme quantity. 2,The plasmid and enzyme activity may not be compatible; optimize the digestion conditions. 3,Ensure the digestion system is thoroughly mixed. 4,The buffer may not be compatible with the enzyme. |
| Q18: | The number of DNA fragments is greater than the theoretical value. |
| A18: | 1,The endonuclease may exhibit star activity. 2,Contamination by other endonucleases; use a new enzyme or buffer. 3,The substrate may contain other DNA impurities; re-extract the plasmid. |
| Q19: | During gel electrophoresis, the bands appear smeared or blurred. |
| A19: | 1,The sample concentration may be too high. 2,High salt ion content. 3,Protein contamination. 4,Degradation of the DNA. 5,Replace the electrophoresis buffer with a fresh one. |
| Q20: | Which column can be used for gel recovery? (Columns from kits for plasmid extraction, gel recovery, genome extraction, PAGE gel recovery) |
| A20: | N1030 centrifugal filtration column is mainly used for filtering large impurities such as gel fragments. N1010/N1012/N1011-A can be used for gel recovery (of plasmids, fragments, etc.). N1011-B is primarily used for genome extraction and not for plasmid extraction (as it can give falsely elevated results). |
| Q21: | In plasmid extraction kits, are all enzymes provided separately? |
| A21: | Yes, because enzymes generally need to be stored at -20°C, they are not included in the kit itself. They are shipped separately in an ice box/ice pack as an accessory. |
| Q22: | Using SN0020 Nuclear Extraction Kit with mouse liver tissue, the homogenate cannot be separated by centrifugation. |
| A22: | Heating the solution slightly can help reduce its viscosity, making centrifugation more effective. |
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