Organic extraction procedures use either phenol or phenol/chloroform mixtures alone [7], [8], [9], [10], [11], or in combination with Triton X-100 [12], or lysozyme [13], [14]. This method eliminates the need for RNase treatment and phenol chloroform extraction if the plasmids are needed only for the restriction digestion. Fields, Pathways Unlike the complete cell disintegration occurring in alkali minipreps, NID plasmid minipreps lead to fragmentation of E.coli cells into small protoplast-like components. 10 l DNA were loaded in the gel. The .gov means its official. DNA restriction and ligation reactions were carried out in NEB1 buffer, but 1 mM ATP was added for the ligation reactions. Be sure not to carry over any phenol during pipetting. 1. Anal Biochem 203(1):169172, McClung JK, Gonzales RA (1989) Purification of plasmid DNA by fast protein liquid chromatography on superose 6 preparative grade. The basic principle of this method is selective alkaline denaturation of high . Biomed Res Int 2017:9306564. https://doi.org/10.1155/2017/9306564, CrossRef Invitrogen, and
19 l 0.25 M EDTA was added and incubation continued for another 5 min. In lanes 3 and 4, all components of the lambda NID extraction buffer were added except lysozyme and Tween 80, respectively. FOIA Note minimal leakage of phage particles in this lane. The mixture was then centrifuged at 40,000 g for 30 min at 0C. We would like to acknowledge Derek Warner and Mike Powers (University of Utah DNA sequencing core) for NID crude lysate sequencing, and Vlas Lezin for technical help for figure preparation, regression analysis and image densitometry. In this report, we have shown that NIDs are highly effective for plasmid DNA isolation and they also extract phage lambda particles. Click through the PLOS taxonomy to find articles in your field. One advantage this system has over other purification methods, such as phenol:chloroform extraction, is its ability to remove most inhibitors of amplification, including very small fragments of DNA. Resuspend the cDNA pellet in 300 L of TEN buffer by pipetting up and down 3040 times. How can I track requests for my plasmids? Adding chloroform, which functionally compensates holin, to an induced S-R+ lysogen (such as UB-61 cells) results in almost instantaneous lysis [33]. Lane 4: 0.5% Tween-80. You will now have plasmid DNA that has been purified away from the bacterial proteins and genomic DNA. Salt concentrations were adjusted to 0.5 M NaCl in all extracts, except the ones loaded in lanes 10 and 1215. Addition of 2050 mM CaCl2 to the extraction buffer reduces extraction of chromosomal DNA and large plasmids, but greatly facilitates formation of cellular debris during sedimentation. There is a problem with the plasmid I received. methods [19], [20], with our NID approach the bacterial suspension remained turbid at room temperature and even for some time after heating, indicating no significant bacterial cell lysis. To your DNA solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of 3 M Na-acetate (pH 4.8). Add one volume of phenol:chloroform:isoamyl alcohol (25:24:1) to your sample, and vortex or shakeby hand thoroughly for approximately 20 seconds. Note: This step causes the bacteria to lyse, releasing their contents, including plasmid DNA, into solution. Many molecular biology techniques require highly purified and concentrated plasmid DNA. Open and invert the tubes on a paper towel to drain them out. Bacterial cells from 2 ml cultures were resuspended in 60 l DEPC/Summerton's sucrose solution (1 l DEPC/500 l Summerton's sucrose solution: 100 mM Tris pH 8.1, 30% sucrose, 100 mM EDTA). Int Microbiol 2:115117, Guerry P, LeBlanc DJ, Falkow S (1973) General method for the isolation of plasmid deoxyribonucleic acid. Interestingly, NID crude lysates could also be used in sequencing reactions because as little as 1 l crude lysate generated reliable sequencing data with trimmed length 783 b (Figure S1A). This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Anal Biochem 76:431441, Ferrus MA, Alonso JL, Amoros I, Hernandez M, Hernandez J (1999) A rapid procedure for the isolation of plasmid DNA from environmental bacteria. for NIDs (Table 1). Biochim Biophys Acta 129:310317, Garger SJ, Griffith OM, Grill LK (1983) Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient. Correspondence to D= re-cut sample. Note: You should see clearly separated layers: Pipet the aqueous DNA layer and place it in a new microfuge tube. We found that 3u KpnI are sufficient to achieve almost complete digestion (Figure 2C). Note: This step removes excess salt from the pellet which can cause problems with some common reactions. Lane 3, isolation according to Clewell and Helinski using Brij-58 and DOX. Dry the cDNA pellet in a Thermo Scientific SpeedVac concentrator for 2 minutes or at room temperature for 510 minutes. Images were recorded using either an Olympus IX81/Metamorph v6.2r6 microscope (100x objective in oil) or a Leica DMR A/Metamorph v6.3r1 (63x objective in oil). Samples were then analyzed and base-called by Applied Biosystems DNA Sequencing Analysis Software V5.2 (Applied Biosystems, Foster City, CA). The phenol-chloroform DNA extraction method is commonly used in molecular genetics and is capable enough to isolate high-quality and high-yield DNA from any biological sample. using Brij-58 and DOX in highly hypertonic salt-sucrose solutions. Arrow indicates irreversibly denatured DNA [1], [25], [29]. J Bacteriol 116:10641066, He M, Wilde A, Kaderbhai MA (1990) A simple single-step procedure for small-scale preparation of Escherichia coli plasmids. The DNA can be stored at -20 degrees C for over eight weeks. A 0.5% NID concentration was more effective for DNA extraction compared to increasing NID concentrations up to 4% (shown for IGEPAL CA-720, Figure 1, lanes 3, 6, 7). Add 200 L of Solution II and invert the tube carefully 5 times to mix the contents. [20], and the classical alkali method according to Birnboim and Doly [1]. Centrifuge at room temperature for 5 minutes at 16,000 g. Carefully remove the upper aqueous phase, and transfer the layer to a fresh tube. However, to ensure that higher salt concentrations did not inhibit enzyme activity, we tested the DNA of the NID procedure with salt sensitive restriction endonucleases, such as SacI and KpnI (Figure 2). An extremely rapid method, INSTA-PREP, has been developed to prepare plasmid DNA from 1 to 3 mL miniprep Escherichia coli bacterial cultures. A plasmid preparation is a method of DNA extraction and purification for plasmid DNA, it is an important step in many molecular biology experiments and is essential for the successful use of plasmids in research and biotechnology. Plasmid purification kits provide the fastest way to obtain a high concentration of clean plasmid DNA. MeSH https://doi.org/10.1101/pdb.prot093351, Flamm NF, Bond HE, Burr HE (1966) Density-gradient centrifugation of DNA in a fixed-angle rotor: a higher order of resolution. However, S+R- lysogens are resistant to chloroform treatment, suggesting that some R gene product is required for chloroform-induced lysis of S-R+ lysogens [34]. Please enable it to take advantage of the complete set of features! Mix by inverting several times. To decrease extraction time, we tested the effects of osmolytes. The water-soluble DNA partitions into the aqueous phase, while the proteins . The DNA can be stored at -20 degrees C for over . Thus, we tested a variety of NIDs, including the NIDs used in Figure 1 as well as Brij-58, Brij-98 (HLB=15), polyoxyethylene tridecyl ether (HLB=14) and TERGITOL TMN 10 (HLB=14.1). Note: This freezing may help the DNA to precipitate. We next compared performance of the pLTM330 plasmid DNA obtained by either NID or alkali miniprep in digestion, ligation and sequencing reactions. Densitometry analysis of plasmid DNA isolated with the different methods is reported. For more information on determining DNA concentration and purity click here. (Optional) Wash the pellet with 70% ethanol. To discuss all the methods of plasmid isolation will require volumes of book space hence in this chapter the most common and trusted protocols used invariably in diffferent laboratories around the world are described. CAS The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Yes DNA usually forms film-like precipitates that adhere well to tube walls and are invisible in isopropanol solutions. Solid-phase in vitro mutagenesis using a plasmid DNA template. Moreover, this method is laborious and time-consuming compared to the other methods available, for example, column-based isolation of DNA. 9 l DNA was also used for every DNA restriction and ligation reaction in 11 l total volume. Phenol-extracted DNA from full lysis samples not subjected to additional purification of either phage particles or phage DNA often degrades or aggregates with impurities upon storage, and it is almost completely resistant to HindIII digestion (G. L. and L.B., personal communication). After drawing 150 l extraction buffer into a pipette tip, the pellet was loosened off the tube wall with the tip without releasing the buffer. Discover a faster, simpler path to publishing in a high-quality journal. Add a denaturing solution to the resuspended bacteria. The boiling lysis method of plasmid isolation is quick and is recommended for the isolation of small plasmids (up to 10 kb). 10 l DNA were loaded. On settling, DNA will remain in the aqueous phase. In: DNA and RNA Isolation Techniques for Non-Experts. Both lysozyme and Tween 80 were critical for phage isolation as in the absence of either one, extraction efficiency decreased markedly compared to the buffer containing all components (Figure 4, lanes 35). A quantitative fluorescent PCR assay was also designed for the detection of MTB DNA. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. 15 l DNA were loaded. Add 1.5 mL of the stock culture to a 1.75 mL microfuge tube. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. Previous investigations have shown that Triton X-100 (HLB 13.5) is about 3 times more effective in solubilizing E.coli cell membrane proteins compared to Tween 20 (HLB 16.7) [35], and high HLB NIDs do not interact easily with the plasma membrane [36]. The phage particles accumulate in host bacteria without lysing them because of a mutation in lambda gene S. Cell lysis and release of phage particles can be achieved by briefly incubating the cells with chloroform [21]. Subsequently, 15 l lysozyme (6 mg/ml in water) was added and incubation continued for 20 min in ice. J Biochem Mol Biol 37:351355, CAS BioTechniques 17:434436, Currier TC, Nester EW (1976) Isolation of covalently closed circular DNA of high molecular weight from bacteria. Google Scholar, Chowdhury EH, Akaike T (2005) Rapid isolation of high quality, multimeric plasmid DNA using zwitterionic detergent. The addition of various osmolytes (sucrose, NaCl, KCl, NH4Cl and NaAc) to the NID solutions decreased the required NID extraction time down to 5 minutes for all NIDs studied (Figure 1, lanes 1014). Lane 1: bacteria were incubated in the isotonic component of the lambda NID extraction buffer, i.e. k and the standard error were estimated by linear regression analysis (Top as a function of n) using the data presented in Table 1, forcing the fitting line to intersect the point of origin. How do I prepare and deposit my plasmids? What do I need to know about the customs and importation process for my country? (1972). Funding: This work was supported by funds to L.B. https://doi.org/10.1016/0022-5193(69)90021-6, CrossRef Arch Biochem Biophys 65:132155, CrossRef No EDTA was included in this buffer. Disclaimer. These data reveal how different plasmid DNA isolation methods affect E. coli cells, and provides evidence for the connection between the bacterial cell morphology induced by a specific procedure and its efficiency. https://doi.org/10.1023/A:1008859632378, Padhye V, York C, Adam B (1997) United States patent US 5658548. 3 Calibrated Step Tablet. Overall, these data demonstrate that NID minipreps save significant time compared to alkali minipreps, and suggest that NID minipreps are particularly suitable for high-throughput applications. Note: This step gets all of the bacteria back into suspension, but within a smaller volume of buffer that is compatible with the next solution. & ORFs. Citation: Lezin G, Kosaka Y, Yost HJ, Kuehn MR, Brunelli L (2011) A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles. Thus, while chloroform is only active with specific genotypes, NIDs are effective irrespectively of the phage genotype. Suspensions were centrifuged at maximum rpm for 10 min or until a tight bacterial pellet was formed. Thus, we isolated pEL04, a low copy number plasmid, using the NID procedure and then assessed digestion. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 445470, Hayes W (1968) The genetics of bacteria and their viruses, 2nd edn. Direct extraction of plasmid DNA from E. coli bacterial cells is achieved by a two-phase solution consisting of phenol-chloroform-isoamyl alcohol and water or buffer with efficient separation of the phases by centrifugation in the presence of the INSTA . In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Plasmid DNA minipreps are fundamental techniques in molecular biology. https://doi.org/10.1016/0003-2697(89)90069-9, Tiselius A, Hjerten S, Levin O (1956) Protein chromatography on calcium phosphate columns. Unauthorized use of these marks is strictly prohibited. Thus, at high sample numbers (when operator-dependent time can replace total time), NID minipreps are 69% more time-efficient compared to alkali minipreps (2.03/1.20 1100). Lane 3: 3u KpnI. If you run into any problems registering, depositing, or ordering please contact us at [emailprotected]
https://doi.org/10.1038/nmeth845, Koo K, Foegeding PM, Swaisgood HE (1998) Isolation of RNA and DNA fragments using diatomaceous earth. Methods Enzymol.100, 243-55. Alkali minipreps require three solutions for completion of plasmid DNA isolation, but NID minipreps require only one. The phenol-chloroform DNA isolation method is also referred to as organic extraction or liquid-liquid extraction. Ligated NID DNA was completely re-cut using SacI (Figure 2B, lanes 3, 6). Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. However, lambda DNA isolated with phenol from NID extracted phage particles shows no degradation or aggregation after 2 month storage at 4C (G. L. and L.B., personal communication). DNA molecular forms were similar when NID crude lysates were immediately precipitated with isopropanol or stored overnight at RT (data not shown). For more information about PLOS Subject Areas, click To further assess the NID procedure, we attempted to extract bacteriophage lambda particles. (B) Plasmid samples digested using 2u SacI for 1 hour (lanes 1,4) were ligated using 0.1 Weiss unit T4 DNA ligase at 15C for 30 minutes (lanes 2,5). Protocols for the Diagnosis of Pig Viral Diseases, https://doi.org/10.1007/978-1-0716-2043-4_6, https://doi.org/10.1016/0022-5193(69)90021-6, https://doi.org/10.1016/0003-2697(81)90473-5, https://doi.org/10.1016/0003-2697(89)90069-9, https://doi.org/10.1016/0147-619x(84)90063-5, https://doi.org/10.1007/s10404-011-0782-9, Tax calculation will be finalised during checkout. Plasmid DNA also usually requires subsequent column purification steps, probably because of SDS contamination. (C): 4 l crude lysates. Resuspend the DNA pellet, or elute the DNA off of the column using water or a neutral buffer such as TE. The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step in many procedures such as cloning, DNA sequencing, transfection, and gene therapy. Brazilian Journal of Medical and Biological Research, 45, 11021111. . Bradley, J., Johnson, D., & Rubenstein, D. (2001). After RNase treatment and phenol/chloroform extraction, the plasmid DNA serves as a good template for sequencing. Ethanol was removed, and the DNA pellet was dissolved in 20-50 l TE buffer. Recent Patents on DNA & Gene Sequences (Discontinued), 5(1), 17. Before (Optional) Place the tube either at -20 C overnight OR -80 C for 30 min OR on dry ice for 5 min. Microscopic analysis showed that the NID procedure fragments E.coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. Clipboard, Search History, and several other advanced features are temporarily unavailable. Here, we have described a one-step, one-solution inexpensive and time-efficient NID-based approach to isolate high quality plasmid DNA and lambda phage particles. CAS If you want to perform plasmid purification without using a kit, you can find a protocol for kit-free plasmid mini-prep at the bottom of this page. No, Is the Subject Area "DNA extraction" applicable to this article? The read length was 800 nucleotides (nt) with 5 ambiguous nt in the first 39 nt (accession number JF804976). 1 Introduction The boiling lysis procedure ( 1) is quick to perform and, therefore, especially suitable for screening large numbers of small-volume Escherichia coli cultures. Enzymatic reactions were performed at 37C for 1 hour if not otherwise specified. Image densitometry/gel quantification analysis was performed using Image J [27]. Thus, the ratio of the variables Talk/Tnid= a+b/Tnid= a+b/nk (see Materials and Methods). When Brij-58 and DOX were used, numerous bacteria maintained a rod-like appearance but some were disrupted (Figure 5, panel B), although the levels of fast and slow-migrating plasmid DNA remained low (Figure 5, lane 3). It is also advisable to add RNAse to the supernatant after step 6 to eliminate RNA contamination. DNA pellets were resuspended in 35 l TE buffer containing 30 g/l RNase A. Approximately 6 hours after heat induction of the lambda prophage and without addition of chloroform, UB-61 cells released some lambda particles in solutions of 5% sucrose and Tris (Figure 4, lane 1), which was defined as leakage of lambda particles. Thus, we assessed E. coli cell morphology and plasmid DNA molecular forms after the NID procedure and the following approaches: isolation using Brij-58 according to Godson and Sinsheimer [18], isolation using Brij-58 and DOX according to Clewell and Helinski [19], isolation using Brij-58 and DOX in highly hypertonic salt-sucrose solutions according to Summerton et al. The particles were precipitated and the lambda DNA was extracted from all samples using the same procedure as described in Materials and Methods. Arrowhead indicates the 400 bp band, and densitometry analysis of this band is reported for lanes 512. However, complete NID miniprep DNA required digestion in the New England Biolabs (NEB) buffer recommended for the specific restriction endonuclease, while all NID crude lysates performed best in NEB buffer 1 (no salt), rendering the crude lysate procedure particularly convenient for most restriction endonuclease digestions. Bacterial media were obtained from Quality Biological (Gaithersburg, MD). In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast saccharomyces: life cycle and inheritance. With the NID miniprep, bacterial cells are not completely disrupted as in the alkali method, but appear to undergo fast re-association as protoplast-like structures which contain most of the chromosomal DNA. This page will discuss the general procedure for purifying plasmid DNA from bacterial culture. Holin, the S gene product, causes lesions (holes) in the cytoplasmic membrane at precise times of the vegetative cycle just before host lysis. Vikas Gupta & Chayanika Das Protocol First Online: 10 May 2022 472 Accesses Part of the Springer Protocols Handbooks book series (SPH) Abstract Plasmid DNA isolation is indivisible step in the development of diagnostic assays based on recombinant proteins and other molecular biology experiments.