How to prepare high quality plasmid? Industry experience from GenScript.
Plasmid preparation from bacterial culture often involves two steps: lysis of the bacteria and purification of plasmid DNA. Alkaline lysis is the most commonly used method for cell lysis for small scale preparation. But multiple methods exist for the plasmid DNA purification step. These method have different advantages and disadvantages and may affect the downstream molecule biology applications.
After the cell lysis and neutralization, the large and less supercoiled chromosomal DNA along with the denatured cellular proteins precipitate out of solution and the plasmid remains soluble. The precipitate can then be easily separated from the plasmid DNA solution by centrifugation or filtration.
However, the plasmid DNA is still mixed with residual cellular proteins and debris, salt, EDTA, and RNase. So a subsequent Purification step is recommended to remove the contaminant from plasmid DNA. The three commonly used methods are:
Adding a phenol/chloroform mixture will dissolve protein and lipid contaminants, leaving the nucleic acids in the aqueous phase. The disadvantages of this method is the risk of phenol or chloroform carry-over into the final sample, which may inhibit downstream enzymatic reactions. In addition, Chloroform and phenol are hazardous to humans. So the method is now less used in industry.
Ethanol precipitation is a popular method for desalting and concentrating DNA. The ethanol can change the structure of DNA, causing it to precipitate out of solution. The soluble fraction (salts and small organic molecules) is discarded, leaving the precipitated plasmid DNA ready for re-suspension. Ethanol precipitation is cheap and effective, but it also precipitates RNA and small ssDNA debris.
Spin column-based nucleic acid purification relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions. The conditions are then changed to elute the purified nucleic acid. This method is commonly used in plasmid mini-prep kits. It is convenient and fast, but is costly than other methods.
Ethanol precipitation and Spin column are the most commonly used method in gene synthesis and cloning industry. More suppliers use Ethanol precipitation because it is cheaper than Spin column method.
How Scientists at GenScript analyze the quality of plasmid?
To understand the quality and yield of the plasmid DNA prepared with the two different method, we tested the plasmids from different aspects.
Using Nanodrop for quantification, we found that the concentration of Spin column prepared plasmid is generally lower than those prepared with Ethanol precipitation. The difference is much larger for low copy number plasmid. Is it because the yield of Spin column method is lower or the DNA concentration measured with Nanodrop is not accurate?
The Nanodrop is an ultraviolet spectrophotometer and measures DNA concentration by the absorbance at 260 nm. But there are too many other substances absorbing at 260 nm so the measurement is not specific. Another commonly used method for DNA quantification is Qubit assays, which is based on the chemistry of fluorochromes. The fluorescent dye specifically binds with the DNA molecules so it can determine the real DNA concentration even for contaminated samples.
We measured the DNA concentration with both the Nanodrop and Qubit system. We found that the plasmid concentration determined by Nanodrop is usually higher than Qubit. This variation between the two methods is larger with Ethanol precipitation. The Qubit concentration for ethanol and column prepared plasmid is very similar. These results indicate that Spin column prepared plasmids are cleaner, but the total yield is at the same level as Ethanol precipitation.
To further determine if the Spin column prepared plasmid is cleaner than ethanol precipitation as this effects downstream cloning reactions, we used the plasmid prepared with both methods to prepare linearized vectors for cloning. We double digested the same amount (Nanodrop concentration) of plasmid prepared and gel purified DNA. We found plasmid prepared with Spin column produced much higher concentration of linearized plasmid than Ethanol precipitation. It further indicates that Spin column prepared plasmid contains less contaminants and is superior for downstream cloning reactions.
At last, we used the plasmid DNA for direct transformation into E. coli competent cells. The plasmid prepared with Spin column produced more colonies with the same Nanodrop amount DNA, further indicating the Spin column prepared DNA contained more real plasmid DNA molecule.
In summary, plasmid prepare with Spin column is cleaner than Ethanol precipitation and is more suitable for cloning and transformation reactions.