Plasmid purification is a rather classical experiment, but the technique is still developing for time- and cost- saving. The critical principle is based on the alkaline lysis method, although the following steps have several variations. The needed purities and/or quantities of DNA depend on researches using isolated plasmids, meaning that more reasonable method can be selected in each experiment. For example, a non-alkaline-lysis method such as boiling method is still available. One of the important steps for purifying plasmid is a removal of RNA. Ribonuclease is usually used for removing RNA from plasmid sample. On the other hand, a kind of salts such as lithium and calcium functions to make RNA as a selective precipitate from DNA-RNA mixture. Based on these backgrounds, the technique to purify plasmid DNA has been discussed.
- plasmid DNA
- chromosomal DNA
- polyethylene glycol
It is said that the word “plasmid” is first proposed by the Nobel Prize winner Joshua Lederberg [1, 2]. Plasmid is an extrachromosomal small circular deoxyribonucleic acid (DNA), which duplicates independently from chromosomal DNA. Although budding yeast and fission yeast can retain plasmid, the host of the plasmid is almost bacteria. This small circular DNA is widely used as DNA vector in molecular biology, biochemistry, biotechnology, cell biology, and so on. It means that plasmid purification/isolation is very fundamental experiment in these research fields, and that this experiment is achieved in almost every laboratories on almost every day.
2. A strategy for purifying plasmid from
In biochemical aspects, to purify plasmid DNA from bacteria is to isolate only plasmid DNA from the mixture of biopolymers such as protein, ribonucleic acid (RNA), chromosomal DNA and plasmid DNA, by which bacteria cell is composed (Figure 1).
A chemical property of protein is totally different from nucleic acids; therefore, it is rather easy to separate nucleic acids and proteins. However, RNA and DNA are very similar molecules from each other. Among them, ribose in RNA is only distinguishable from deoxyribose in DNA by one hydroxyl group (−OH) at its structure. Furthermore, chromosomal DNA and plasmid DNA is both deoxyribonucleic acids that have the same chemical properties. Chromosomal DNA in almost all bacteria is circular, so is also plasmid. The distinguishable difference of them is only their size: plasmid DNA (~10 kilo base pairs) is much smaller than chromosomal DNA (4.6 million base pairs in
To purify plasmid DNA of high quantity, culture condition, or media for
3. Very easy and simple way of plasmid preparation: boiling method
A very simple manipulation steps enable us to recover plasmid DNA from
Interestingly, the pellet is rather moisty and easily removed by using toothpick and piercing it. This feature contributes to an easy handling of the experiment: we can achieve this plasmid extraction in only one tube from the start point to the end of the experiment. Therefore, boiling method has very convenient for handling many samples at a time. The modified boiling method, in which a concentrated STET solution is directly added to LB medium in which
The major disadvantage of the boiling method is that RNA is not removed in the principle of the boiling method and that the chromosomal DNA of
One substitution of the boiling method for insert check is colony polymerase chain reaction (PCR), directly adding
4. The definitive principle for plasmid isolation: denaturation of DNA double-strand by alkaline lysis
To purify plasmid from
The principle of the alkaline lysis method is a kind of magic. After suspending the
In early days, original protocol of alkaline lysis method used sodium acetate as a salt in solution III . However, now potassium acetate is substituted for the major agent for solution III than sodium acetate. Potassium ion binds to dodecyl sulfate ion and forms potassium dodecyl sulfate (PDS). PDS is highly insoluble salt, which is made by adding solution III in the alkaline lysis sample. The PDS also plays a seed for insoluble debris, with which insoluble proteins and chromosomal DNA are co-precipitated. It is the great advantage that alkaline lysis method enables us to prevent protein and chromosomal DNA from plasmid at the same step.
One point we have to keep in mind is that supercoiled (closed circular) plasmid DNA is converted into nicked, relaxed (open circular) DNA by alkaline incubation. Thus, we have to keep the incubation time at solution II as in the instruction, and not to incubate the sample with solution II for a long time. Besides, RNA is not removed in a series of alkaline lysis method for plasmid purification (Figure 3). RNA is partially hydrolyzed by solution II but remains with the plasmid DNA at the final step. Therefore, a huge amount of RNA contamination is in the final plasmid DNA sample. Principally, only isolating plasmid DNA by alkaline lysis is inadequate for purifying high-quality plasmid DNA, and several schemes for further purification of the plasmid DNA, especially for removing RNA, should be needed.
Anyhow, alkaline lysis method has been the definitive way to initially purify the plasmid DNA.
5. RNA removal from plasmid sample
Neither alkaline lysis method nor boiling method does not isolate plasmid DNA from RNA mixture. An extra step for removing RNA is needed for further purification of plasmid DNA.
5.1. Ribonuclease (RNase)
Basically, to remove RNA (not to separate intact RNA) from DNA-RNA mixed solution is very easy: Only to add ribonuclease (RNase) to the solution enables us to completely digest RNA. Even when RNase is added to the solution I in the course of alkaline lysis method, RNA is completely digested in the finally corrected plasmid sample (see Figure 3). It sounds very strange that RNase in the solution I digest RNA, because the function of solution I is only to suspend the
Irresponsible usage of RNase often contaminates the laboratory. Therefore, after incubating plasmid sample with RNase, the complete inactivation/removal of RNase should be needed.
5.2. Removal of RNase by phenol/chloroform extraction
Phenol or phenol/chloroform is well known as a protein denaturant. RNase is also inactivated by such as denaturant. Because RNase is very stable, repeating steps of phenol or phenol/chloroform extraction is effective for the complete removal of RNase. On the other hand, this organic reagent often inhibits enzyme activities, once contaminated with the nucleic acids samples. Besides, it is very convincing that phenol or phenol/chloroform have toxicity to cells, resulting in a decrease of transfection efficiency to cultured cells, and so on.
5.3. Salts as an agent for nucleic acid precipitation
It is known that a certain salts selectively precipitate nucleic acids. These salts can be applied to plasmid DNA purification.
5.3.1. Lithium chloride
Lithium chloride (LiCl) at the final concentration of 2.5 M enables us to selectively precipitate RNA. In this condition, RNA makes a pellet by centrifugation, but not DNA. Although low-molecular-weight RNA fragment is not precipitated and remains with plasmid DNA, it is often an adequate quality for using the plasmid in the following experiments, as long as the low-molecular-weight RNA makes critical disturbance for the experiment.
In isolating plasmid DNA by boiling method, Adding LiCl to STET is a better way to do the experiment (see Figure 2). After boiling step, centrifugation makes insoluble debris, together with RNA precipitation by the function of LiCl. Therefore, one-step centrifuge is enough for removing protein and RNA. Rather low-molecular-weight RNA still remains in the solution, but normally this RNA does not disturb or inhibit the activity of restriction enzyme and so on. The insoluble pellet in the boiling method with LiCl is like a chewing gum, and is easily removed by picking with toothpick. Therefore, the combination of boiling method with LiCl is a very reasonable choice. One minor point is that LiCl is rather expensive than Calcium chloride (CaCl2).
5.3.2. Calcium chloride
Calcium chloride (CaCl2) is an inexpensive reagent. It is also known to precipitate RNA at the concentration of around 1 M, but DNA is not precipitated in this condition . Thus, this reagent also works in the plasmid purification process like LiCl. However, it is a luck of luckiness that when CaCl2 is added instead of LiCl in the boiling method, insoluble debris forms crumbly. This means that we cannot pick the debris up by using toothpick, and that we need another tube to transfer the supernatant after centrifugation. This disturbs a merit of the boiling method using only one tube all over the manipulations.
5.4. Polyethylene glycol precipitation of DNA
Polyethylene glycol (PEG) can be used to precipitate DNA . It is also reported that the size of precipitated DNA is controllable by the concentration of PEG . The principle of PEG precipitation is the same as alcohol precipitation, such us ethanol and/or isopropanol. This compound selectively precipitates DNA. Especially, low-molecular-weight RNA, such as transfer RNA, is not precipitated by PEG. There are so many products of PEG, according to their average molecular weights. Generally, PEG #3000, #4000, or #6000 has similar properties for DNA precipitation. Interestingly, to the contrast of that LiCl and CaCl2 do not precipitate low-molecular-weight RNA, the size between precipitated RNA by salts and non-precipitated RNA by PEG precipitation are complementary in the RNA length from each other.
5.5. Cesium chloride ultracentrifuge
Cesium chloride (CsCl) ultracentrifuge method  does not require RNase. It means that phenol/chloroform extraction is not needed in the experiment, so plasmid DNA purified by this method is suitable for the almost all the biochemical experiment. That is, we can apply the plasmid DNA isolated by this method to transfection of the cultured cell and so on.
An ultrapure grade of plasmid is obtained in this method, although a special expensive ultracentrifuge is required for equipment. Moreover, very long time (almost overnight) for centrifuge is needed, and ethidium bromide (EtBr) at a very high concentration (final 800 μg/mL, this is 8000 times higher concentration than agarose gel electrophoresis) is used. EtBr is widely known as a mutagen, and highly concentrated EtBr should be unwanted to handle, if possible. EtBr intercalates to the double-strand of DNA. When this compound is intercalated with DNA, the double-strand of plasmid DNA changes to the slightly unwound form. This affects the sedimentation coefficient of nucleic acids in 10% CsCl solution. Therefore, supercoiled plasmid DNA makes a single sharp band in the tube after ultracentrifugation (200,000×
CsCl ultracentrifuge method costs expensive because CsCl is an expensive reagent. Moreover, this experiment is time-consuming, hazardous, and difficult. On the contrary, once succeeded, we can obtain a large amount of ultrapure plasmid DNA. This method can even separate ccDNA from ocDNA, trusting the super high quality of plasmid DNA.
The important fact is that CsCl method is a kind of post-manipulation of alkaline lysis method. That is, alkaline lysis method is such a universal method that it works well as an initial step of CsCl method.
6. Standard plasmid purification method in recent days
Two major kit for plasmid purification is available in the market (Figure 4). Both use a basic alkaline lysis method for initial steps, and also uses RNase for RNA removal. A feature of the recent plasmid isolation methods is that they do not go through phenol/chloroform extraction after RNase treatment. Organic solvents are often harmful to cultured cell and so on, so avoiding this reagent in the steps of plasmid purification is a reasonable choice.
6.1. Qiagen column kit
Diethyl-aminoethyl (DEAE) group has positive charges; therefore DEAE-resin is often used to ion-exchange chromatography. DNA also has negative charges, so it binds to DEAE-resin under a certain pH or salt concentration. However, DNA purification by using DEAE was restricted to the recovery step from excised agarose gel and so on, because the binding property of nucleic acids to DEAE is rather broad and weak. Plasmid purification by anion-exchange chromatography has been reported , but it will be much better using the column as disposable to avoid contamination of samples. Therefore, DEAE was not seemed to be applicable for plasmid purification in a daily experiment. Qiagen column is famous for its ultrapure quality of purified plasmid DNA, and the principle is ion-exchange column chromatography. The precise information of Qiagen resin is confidential, but basically, it is known that the column consists of a highly condensed anion-exchange group resin . In this experiment, the agent to remove RNA is not an ion-exchange resin, but RNase. A high concentration of RNase is added in the initial step of solution I. During Qiagen chromatography steps, the solutions go through a column in a free fall, so centrifuge step is not required at each step. However, after elution of the plasmid DNA from the resin, the concentration of plasmid DNA may be low, which is inadequate for the following experiment. Therefore, on Qiagen kit, ethanol precipitation is often needed to the eluted solution for concentrating plasmid. It seems that Qiagen columns are suitable for purifying plasmid of mini or maxi scale because the column is rather expensive. Besides, it is not easy to manipulate for multiple open columns at a time.
6.2. Boom’s method (silica-membrane kit)
Boom’s method is based on a paper and patent by Boom et al. , although the principle is widely known as a biochemical property of nucleic acids. The principle of Boom’s method is that glass powder or diatomaceous earth (the main ingredient is SiO2) adsorbs nucleic acids in a chaotropic condition , whereas proteins are not. In Boom’s method, guanidine hydrochloride or guanidine thiocyanate is often used for chaotropic agent. This adsorption is reversibly eluted by pure water. Hydrophobic condition keeps the adsorption of DNA to SiO2, so washing glass powder which adsorbs nucleic acids by 70% ethanol contributes to a high purity of plasmid DNA. The original Boom’s method uses grass or diatomaceous earth powder as binding agent, and each step for binding, washing, and eluting is achieved as batch technique (simply centrifuging and discarding the solution). Batch chromatography is very simple method and easy to handle, although pipetting and discarding each solutions makes the experiment rather complicating for manipulating many samples at a time. On the other hand, the commercial kit supplies a column with a silica membrane filter, which is set to 1.5 mL microtube. Once the solution is applied to column, centrifuge step forces the solution go through the column. Therefore, each steps for binding, washing, and eluting needs only several seconds (as much as 1 minute) for centrifugation. Actually, commercial kit of such a silica membrane filter is very easy and useful for handling.
6.2.1. Homemade reagents for Boom’s method
The commercial kit supplies their reagents with the column, but the compositions of these reagents are always confidential . On the other hand, the principle of these kits seems almost the same, based on the DNA adsorption to silica matrix in chaotropic solution. Although it is quite natural to assume that each kit has its own special reagents, homemade solutions based on the original paper are generally available. When applied homemade reagents to commercial silica membrane column, quality and quantity of purified plasmid are almost the same as the commercial kit . It means that these solutions are available by DIY and columns are not still waste, even when reagents in the commercial kit box are expired and out of use.
A modified reagent and modified protocol are also reported to increase the recovery efficiency of the plasmid DNA by commercial column . On the other hand, not silica particles but Zirconium dioxide (ZrO2, zirconia) has also been reported as an adsorbent of DNA .
6.2.2. Ultra-mini-scale purification
The chaotropic agent is a key factor in Boom’s method, so guanidium salt such as GuHCl should play an important role for plasmid DNA purification in Boom’s method. But it is reported that guanidium salt is not actually needed for nucleic acids to be adsorbed to silica particles. The other reagent such as high-concentration NaCl also works as chaotropic agent . More surprisingly, a high concentration of the salt in solution III of alkaline lysis method seems already adequate for making the solution to chaotropic condition . It means that adding guanidium for DNA adsorption can be skipped. To cut steps in the experiment has many advantages, especially for handling many samples at a time. Therefore, purifying plasmid samples in 96-well plate without guanidine chaotropic condition is proposed , in which method small scale and many samples at a time.
7. 55-minute method
Based on the alkaline lysis method, we developed a new plasmid purification method, which ends within 1 hour and does not need RNase (Figure 5) . The principle of this method is a combination of the alkaline lysis method, CaCl2 precipitation, and PEG precipitation. Although a sequential combination of these precipitations was already reported , our new invention is that we developed a new composition of solution III. This “super solution III” contains CaCl2 to the standard solution III (Solution III: 5 M CaCl2: H2O = 2:2:1), which makes not only protein and genomic DNA debris but also a pellet of RNA in the debris. After centrifuging and recovering a supernatant, a standard PEG precipitation makes a plasmid DNA pellet and removes small RNA, which was not precipitated at the super solution III step. After all, only plasmid DNA remains in the final solution. Actually, this method needs totally 55 minutes from collecting
In a course of doing plasmid purification for every day, I noticed several tips for the experiment.
Alkaline denaturation/renaturation steps are so sophisticated that it is the definitive method for plasmid purification. None of another method will take over the alkaline lysis method. However, RNA is not removed in alkaline lysis method, so RNA removal steps should be applied in a course of plasmid isolation by alkaline lysis method.
RNase is an easy choice to remove RNA, but should be completely removed after RNA digestion. One of the solutions is phenol/chloroform protein extraction, but phenol/chloroform may play a troublesome factor. Only a slight contamination of this reagent inhibits the activities of several enzymes and disturbs biochemical experiments. It also has toxicity to the cells, also disturbing transfection experiments. In other words, eliminating phenol/chloroform step in plasmid purification is the key point to trust its purity.
Qiagen kits and Silica-membrane kits are actually the extra steps after alkaline lysis method. These kits need RNase for RNA digest. In other words, they work as RNase remover from the solution.
RNase completely digests unwanted RNA from the plasmid sample. But this enzyme is very stable and very hard to inactivate, even disturbing RNA experiment in the laboratory. Moreover, RNase is usually isolated from animals such as bovine, which may induce allergy to the human in gene therapy .
Based on these tips, we developed a new composition of solution III on alkaline lysis method, which enables us to purify plasmid DNA without adding of RNase. This method does not need any special columns or resins, but plasmid DNA purified by this method has enough quality for applying transfection to the cultured cell, injection into the nematode, and so on. Our result indicates that plasmid DNA purification without phenol/chloroform extraction is a great advantage for the quality of purified plasmid DNA.
I thank Ms. Haruka Yano (Tokai University Graduate School) for technical suggestions.
Conflict of interest
The author has no conflicts of interest directly relevant to the content of this article.
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Lederberg J. Cell genetics and hereditary symbiosis. Physiological Reviews. 1952; 32:403-430. DOI: 10.1152/physrev.19126.96.36.1993
Cohen SN. DNA cloning: A personal view after 40 years. Proceedings of the National Academy of Sciences of the United States of America. 2013; 110:15521-15529. DOI: 10.1073/pnas.1313397110
Wood WN, Smith KD, Ream JA, Lewis LK. Enhancing yields of low and single copy number plasmid DNAs from Escherichia colicells. Journal of Microbiological Methods. 2017; 133:46-51. DOI: 10.1016/j.mimet.2016.12.016
Holmes DS, Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Analytical Biochemistry. 1981; 114:193-197
Elkin CJ, Richardson PM, Fourcade HM, Hammon NM, Pollard MJ, Predki PF, Glavina T,Hawkins TL. High-throughput plasmid purification for capillary sequencing. Genome Research. 2001; 11:1269-1274. DOI: 10.1101/gr.167801
Peng X, Yu KQ, Deng GH, Jiang YX, Wang Y, Zhang GX, Zhou HW. Comparison of direct boiling method with commercial kits for extracting fecal microbiome DNA by Illumina sequencing of 16S rRNA tags. Journal of Microbiological Methods. 2013; 95:455-462. DOI: 10.1016/j.mimet.2013.07.015
Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research. 1979; 7:1513-1523
Eon-Duval A, Gumbs K, Ellett C. Precipitation of RNA impurities with high salt in a plasmid DNA purification process: Use of experimental design to determine reaction conditions. Biotechnology and Bioengineering. 2003; 83:544-553. DOI: 10.1002/bit.10704
Paithankar KR, Prasad KS. Precipitation of DNA by polyethylene glycol and ethanol. Nucleic Acids Research. 1991; 19:1346
Hartley JL, Bowen H. PEG precipitation for selective removal of small DNA fragments. Focus. 1996; 18:27
Green MR, Sambrook J. Preparation of plasmid dna by alkaline lysis with sodium dodecyl sulfate: Maxipreps. Cold Spring Harbor Protocols. 2018. DOI: 10.1101/pdb.prot093351
Grunwald AG, Shields MS. Plasmid purification using membrane-based anion-exchange chromatography. Analytical Biochemistry. 2001; 296:138-141. DOI: 10.1006/abio.2001.5197
QIAGEN Plasmid Purification Handbook. 2nd ed. Hilden, Germany: QIAGEN; 2003. pp. 76-77
Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheimvan Dillen PM, van der Noordaa J.Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology. 1990; 28:495-503
Marko MA, Chipperfield R, Birnboim HC. A procedure for the large scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Analytical Biochemistry. 1982; 121:382-387
GenElute™ Plasmid Miniprep Kit. St. Louis, MO: Sigma-Aldrich; 2010
Sasagawa N, Yano H. Plasmid DNA Purification using specialized reagents. Proceedings of the School of Engineering of Tokai University. 2017; 57:1-4
Pronobis MI, Deuitch N, Peifer M. The Miraprep: A protocol that uses a Miniprep kit and provides Maxiprep yields. PLoS One. 2016; 11:e0160509. DOI: 10.1371/journal.pone.0160509
Saraji M, Yousefi S, Talebi M. Plasmid DNA purification by zirconia magnetic nanocomposite. Analytical Biochemistry. 2017; 539:33-38. DOI: 10.1016/j.ab.2017.10.004
Lakshmi R, Baskar V, Ranga U. Extraction of superiorquality plasmid DNA by a combination of modified alkaline lysis and silica matrix. Analytical Biochemistry. 1999; 272:109-112. DOI: 10.1006/abio.1999.4125
Neudecker F, Grimm S. High-throughput method for isolating plasmid DNA with reduced lipopolysaccharide content. BioTechniques. 2000; 28:106-108
Sasagawa N, Koebis M, Yonemura Y, Mitsuhashi H, Ishiura S. A high-salinity solution with calcium chloride enables RNase-free, easy plasmid isolation within 55 minutes. BioScience Trends. 2013; 7:270-275
Sauer ML, Kollars B, Geraets R, Sutton F. Sequential CaCl2, polyethylene glycol precipitation for RNase-free plasmid DNA isolation. Analytical Biochemistry. 2008; 380:310-314. DOI: 10.1016/j.ab.2008.05.044
Eon-Duval A, MacDuff RH, Fisher CA, Harris MJ, Brook C. Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process. Analytical Biochemistry. 2003; 316:66-73