lismsid preparation is a method used 0 extract and purify plasmid DNA, Methods developed to purify jnid DNA from bacteria generally involve harvesting and alkaline lysis of the bacteria and precipitation jomosomal DNA and protein, followed by purification of the plasmid DNA. Here, we describe the gUji:preparation of plasmid DNA by a rapid small-scale method, adapted for Listeria: monocytogenes. { quality of plasmid DNA isolated using this method is sufficient for analytical purposes but may be led for further downstream analysis, Electrophoretic separation of the resultant lysate allows Jusions to be made on the presence, number, copy number, and size of the plasmids in the analyzed Plasmids naturally exist in virtually all bacterial species. These small, circular, double-stranded DNA molecules are distinct from the cel’s chromosomal DNA, replicate independently of the genome, and are stably inherited. Plasmids generally only account for a small fraction of the bacterial genome, and their lengths can vary in size from a few kilobases to many hundred kilobases [1]. Many copies of plasmids of lower molecular weight may exist in a cell, whereas fewer copies of larger plasmids are found, Plasmids are of significant importance in the prokaryotic world as they often provide the bac- terial cell with a genetic advantage [2], such as antibiotic resistance or other traits [3, 4]. In addition, plasmids are a key vector for hori- zontal gene transfer in bacteria through conjugation [1, 5]. Given their importance for conferring adaptive traits to the host organism, interest in stadying and sequencing plasmids is growing. For plasmid isolation, bacterial cells are generally growa to late logarithmic or early stationary phase. Lysozyme is used to weaken the cell wall, thus achieving easier enzymatic lysis. Like chromosomes, Jordan ata (os), Listeria monocytogenes: Mathods ard Protocols, Methods in MetcGuar Biology, vol. 1187, fp) 10soa78-1-4a30-0709.815, © Springer Senco: Business Meda New York 2014 181Aidan Casey ang tins Meauatie plasmids generally exist in a covateny closed cigcular (supercoile.!) form, and most isolation procedures are based on this fact. Atier @ gentle alkaline lysis, plasmis can be found in linear, open circle multiple-supercoiled form which allows for easier discrimination, Since DNA is very sensitive to mechanical stress, all mixing steps | during and after cell lysis should be performed carefally by invert ing the tubes to prevent shearing. At this point, itis necessary t¢ remove all intracellular macromolecules in order to enrich an Purify the plasmid DNA. Adding to sodium acetate allows the cit: cular DNA to renature, while sheared cellular DNA remains dena- tured as ssDNA. This ssDNA is then precipitated along with otheti: large molecules, which allow the separation of the contaminant from the plasmid DNA. Phenol-chloroform extractions arc uscd is this instance, followed by ethanol precipitation of the DNA a RNase treatment. Analysis of plasmid DNA is achieved by horizon: tal electrophoresis at 4 °C at low voltage. This mini-preparation method generally yields between 5 and 10 pg of relatively pi plasmid DNA which is generally sufficient for a number of uses, such as strain characterization, in vitro transcription or translations and restriction digestion. For other downstream applications such, as ligation, transformation, or sequencing, scale-up and addition purification techniques may be required which will yield higher concentrations of plasmid DNA. ‘To 50 mL water, add 12.5 g sucrose to give a 25 % sucrose solution. ‘The resulting solution is autoclaved at 121 °C for 15 min. Add 1.5 g of lysozyme to the autoclaved solution and stir thoroughly to dissolve, yielding a 25 % sucrose solution containing 30 mg/mL lysozyme. Once the lysozyme is fully dissolved in solution, sterilize the solution once more by passing it through a 0.45 im filter into a sterile container. a ‘To 200 mL of HO, L6 g of NaOH is added, stirring to dissolve, to yield a concentration of NaOHI of 0.2 N (see Note 1). The solution is then autoclaved at 121 °C for 15 min, To the autoclaved 4 solution, add 6 g of sodium dodecyl sulphate (SDS) to give a final concentration of 3% w/v SDS in 0.2 N NaOH. To 100 mL of H,O, add 24.61 g of anhydrous sodium acetate to | give a concentration of 3 M sodium acetate in solution, Adjust the pH of the solution to 4.8 by adding a small quantity of concentrated hydrochloric acid (see Note 2). Once the pH has been adjusted to 4 the desired level, sterilize the solution by passing it through a 0.45 um filter. Put the solution on ice prior to use for the plasmid isolation protocol.ress, all mixing 48 d carefully by ing | ing, itis necess | order to enti | cetate allows on of the DNA fi cchieved by hori sis mini-prepar £ of relatively [2 for 15 min, {stir thoroughly: aining 30 mg/mf | | yofconcentrated. been adjusted to ing it through a ‘ for the plasmid anid Analysis of Plasivic) GNA isleria monocytogenes Yo $0 mL. of HO, add 28.9! ¢ of ammonium acetate powder in order io make up 2 solution with a final concentration of 7.5 M. Once acid has been fully dissolved, sterilize the solution by passing it through a 0.45 jum filter. Firstly, make up 50x TAE buller by weighing out into a large conical flask 242 g of Tris (Trizma buffer from Sigma), $7.1 mL of glacial acetic acid, and 18.6 g of EDTA. Make up to 1 L with distilled ‘Water, stirring to dissolve, ‘Transfer into five separate 200 mL bottles. The buffer is then autoclaved at 121 °C for 15 min. Dilute 50x TAE buffer down to 1x by adding 20 mL of 50x butler to 980 mL of sterile water. The final concentrations are as follows: For 50x: Tris—2 M, glacial acetic acid—1 M, EDTA— 50 mM. For 1x: Tris—40 mM, glacial acetic acid~-20 mM, EDTA—I mM. Once 1 L of Lx TAE buffer is made up, add 20 yl. of ethidium bromide and mix by inverting (see Note 3). | sodium acetate to 3 lution, Adjust the 1. Picking a colony from a previously streaked agar plate contain- ing the desired strain of Listeri monocytagenes, resuspend the bacteria in 10 mL of enrichment broth (see Note 4) and incu- bate overnight at 37 °C. 2. Following the resulting overnight incubation, transfer 100 iL, of the bacterial culture into 5 mL of sterile media and incubate for 4-5 hat 37 °C. 3. Once sufficiently turbid, centrifuge the culture at 2,500 for 10 min, and discard the supernatant. The remaining bacterial pellet is then used in the subsequent plasmid isolation protocol (see Note 5). 1. Resuspend cach bacterial pelict in 200 pL of the 25 %sucrose containing 30 mg/mL lysozyme solution (ser Note 6), and for each, transfer the 200 pL solution to a sterile 1.5 mL Eppendorf tube. 2, Incubate Eppendorf tubes at 37 °C for 20-25 min. 3. To each Eppendorf, add 400 wL of the alkaline SDS solution (see Note 7), and mix thoroughly by inverting the tube several times. 4. Incubate Eppendorf tubes at room temperature for 7 min. 5. Vortex each sample for 30 s (see Note 8). 6. To each sample, add 300 pL of ice-cold 3 M sodium acetate. ‘Mix samples immediately by inversion, before centrifuging the Eppendorf tubes at 18,500 xg for 15 min and at a temperature of 4°0.194 Aidan Cas McAuliffe ?, Transfer 700-800 pit. of the resulting supernatant from cach, tube to another sterile 1.5 ml. Eppendorf tabe, and discard’ the pelle. To each of these, add 650 |lL of isopropanol (room temperature). Centrifuge these samples once more at 18,500 xg for 15 mine and ata temperature of 4 °C. ‘Once centzifagation is complete, remove all liquid from acl of the tubes (see Note 9), and resuspend the remaining pelle in 320 pL of sdH,0. When pellet is completely dissolved, add 200 pL of 7.5 ammonium acetate to each tube and mix by inversion. . To cach tube, add 350 iL of phenol/chloroform, and im tubes, Centrifuge samples at 18,500xg for § min and at temperature, . Transfer the upper phase (approximately 300 iL) from ead tube into a sterile Eppendorf tube, and to each, add 1 mb g 100 % ethanol (stored at a temperature of -20 °C) . Immediately centrifuge samples at 18,500xg for 15 min and a temperature of 4 °C. Following centrifugation, pour off the 100 % ethanol, wash the remaining pellets with 200 pL of 70 % ethan (see Note 10). i - Completely remove the ethanol from the tubes, and dissol each pellet in 40 UL of H.O containing 0.1 mg/ml. of RNagg . Store samples at 4 °C for subsequent running on an agarose g ‘Make up a 0.7 % agarose gel by weighing out 1.4 g of agarosd and suspending it in 200 mL of 1x TAE buffes-(containi ethidium bromide). . Microwave for $-6 min to fully dissolve agarose, and allow j to temper at room temperature on worktop bench, bet pouring into a previously set up gel cast unit. Insert a 10- comb immediately without introducing air bubbles. . Allow the gel to set and solidify for 10-15 min. ‘4. Prepare each of the plasmid DNA samples for running on th gel by adding 10 pL of plasmid DNA to 10 pl of 2x load dye (final concentration of 1x loading dye). ‘Transfer the gel into the running unit, and carefully remo¥ the comb. . Pipette the 20 iL solutions for’ each sample into each of wells.inversion. oform, and is 90 iL) from, ach, add 1 10°C). Extraction and Anelygie of Plaamic ONA trom Listeria monocytogenes 7, Into a weil at either end of the loaded samples, add 4 pL of 2 1 kb ladder. 8. Run the gel overnight at a temperature of 4 °C and at a voltage of 45 V. 9. ‘Take an image of gel under UV light in order to visually con- firm the isolation of plasmid DNA. Successfully isolated plas- mid DNA should appear in one, or several, clear and concise ‘bands on the agarose gel. 1. SDS powders harmful to the respiratory system ifinhialed. When ‘weighing out powder, ensure that necessary precautions (such as weighing powder inside a fame hood) are taken for safety. 2. Addition of concentrated hydrochloric acid to reagents when adjusting the pH should be done in a laminar flow hood as fumes from concentrated hydrochloric acid may be harmful. 43. Bthidium bromide is mutagenic and carcinogenic and should be handled with care at all times. Protective gloves should be worn when working with this chemical 4. Enrichment broth such as tryptic soy broth or brain-heart infusion broth is sufficient for growth of the Listeria monocyro- _gencs strains in preparation for the plasmid isolation protocol. 5. Itis also recommended that a bacterial strain be grown in tan- dem, of which the plasmid content is already known, to use as a positive control for the experiment. If possible, use a strain with known plasmid sizes, and which have a combination of large and small plasmids, so that this strain may also be used as a reference ladder when run out on an agarose gel. 6. Following addition of lysozyme solution to bacteria, the time of incubation required to achieve lysis of the cells depends on the nature of the bacteria of interest. For bacteria such as Lactococcus, the incubation time required is generally between 10 and 15 min. For Listeria, these times need to be increased to ensure that fall lysis is achieved. 7. Alkaline SDS solution should be tempered at 40 °C prior to use, as storage of the solution at cooler temperatures can cause the SDS to precipitate out of solution. 8: Vortexing of the samples is optional. While vortexing of sam- ples is not generally recommended at any point during the pro- tocol, in this instance, it should have the effect of shearing, genomic DNA while not affecting the closed plasmid DNA. 9. After the 15-min centrifugation in isopropanol, resulting sam- ples should consist of a clear supernatant with a white pellet atNive MeAuitte the bottom of the tube. Ensure that all of the supernatant bas been removed befure resuspending the pellet in dsHLO. (f necessary, spin the tubes several times on a small bench-top. 20 iL tips. The more isopropanol that remains in the tubeys the more difficult the sample will be to resuspend in water, Dissolving of the pellet in dsH3O can take anywhere from 2 10 min, and incubating the tubes at 37 °C may help the pellets dissolve faster. When washing the pellets with 70 %, be careful to ensure th the pellet does not become dislodged from the bottom the tube References L.Smillie C et al (2010) Mobility of plasmids, 4,PoyareSalmeron C et al (1990) Teansferabig Microbiol Mol Biol Rev 74:434-452 plasmid-mediated ancbiotic resistance in Lister 2,Ratani $8 et al (2012) Heavy metal and disinfee- monocytogenes. Lancet 335 ‘tant resistance of Listeria monocytysenes from 1422-146 foods and food processing plants. Appl Environ 5. Hlarcison E, Brockhusst MA (2012) Plasmi ‘Microbiol 78:6938..6945 mediated horizontal gene transfer is a coevol 3. Kuenne C et al (2010) Comparative analysis of tionary process. Trends Microbiol 2 plismidsin the genus Listeria. PLoSOneS:e12511 262-267