Application Note: AN-LECF- Traditional Methods for CsCl Isolation PLASDNA-0408 of DNA by Ultracentrifugation

Stephanie R. Noles, PhD, Thermo Fisher Scientific, Laboratory Equipment Division, Associate Product Manager

Introduction greater laboratory productivity and a final concentration of 1mg/mL Key Words are pieces of extra-chromo- instrumentation and rotor life. lysozyme • Density Gradient somal DNA found in . They This application briefing will de- • Remove cellular debris by cen- Centrifugation exist in double-stranded, closed cir- scribe multiple procedures for CsCl trifuging viscous solution at cular form, and they carry genes isolation of plasmid DNA using 33,000 x g (15,000rpm) for 15 • Cesium Chloride which convey a variety of traits to three Thermo Scientific ultraspeed minutes at 20°C in a Thermo Gradients (CsCl) the organism, such as antibiotic re- centrifuge rotors: StepSaver™ Scientific RC-6 Plus with the • Ultracentrifugation sistance and the production of viru- 65V13, TFT-80.2 and TFT-80.4. SA-600 rotor lence factors. Because plasmids are • Precipitate nucleic acids by mixing • TFT80.2, TFT80.4, easy to handle and manipulate, they Reducing Spin Times for the CsCl supernatant with an equal volume Isolation of Plasmid DNA Stepsaver 65V13 are utilized extensively in the cloning of isopropanol and freeze at -20°C Dr. Mark W. Schwartz and Rosanna for 1 hour • Plasmid DNA of genes and in the study of molecu- A Fischer Purification lar biology. To clone a gene using a • Pellet precipitated DNA by cen- plasmid as either the vector or the trifuging at 21,000 x g (12,000 source of the gene to be cloned (e.g. rpm) for 15 minutes at 4°C in the antibiotic resistance), plasmids of Sorvall RC-6 Plus with the SA-600 high purity must be obtained. A va- rotor riety of procedures exist for the iso- • Resuspend pellet in 4mL 0.1M Tris lation and characterization of pH 8.0 plasmid DNA. Generally, procedures • Incubate for 30 minutes at 37°C involve growing the organism, har- with 2mg/mL RNase vesting cell material, lysing cells • Extract solution by the addition of using a detergent solution with or equal volume of phenol without lysozyme, precipitating cell • Follow with two extractions with debris and chromosomal material In the following application brief, 24:1 chloroform:iso-amyl alcohol and finally recovering plasmid DNA the use of the step-run feature in • Obtain precipitated DNA by by precipitation. One traditional Thermo Scientific Sorvall® ultra- adding three times the volume of DNA purification technique in use speed centrifuges with the Stepsaver and freeze at -80°C for 1 since the 1950s is cesium chloride 65V13 vertical rotor to reduce spin hour (CsCl)/ethidium bromide (EtBr) cen- times during the preparation of plas- • Collect plasmid by centrifuging at trifugation (Radloff et al., 1967; mid DNA by CsCl isolation is de- 33,000 x g (15,000 rpm) for 15 Maniatis et al., 1982; and Meselson scribed. Several procedures minutes at 4°C in a RC-6 Plus et al., 1957). Although the funda- (standard protocol, step run, and with the SA-600 rotor mentals of the technique have re- layered sample) used for isolating • Resuspend pellet in 500µL of 0.1M mained unchanged, advances in the plasmid pRH43 from Es- Tris, 0.5M EDTA, pH 8.0 (TE) rotor design and an increased under- cherichia coli (E. coli) are described. buffer and store at 4°C standing of centrifugal theory have led to better control over protocol Procedure Multiple Methods for Density Gradient Centrifugation conditions and significantly reduced DNA Preparation Standard Protocol spin times (Flamm et al.,1966). Plas- • Grow E.coli colony harboring plas- • Bring DNA sample (500µL) to mid isolation in CsCl gradients has mid pRH43 to an OD600 = 0.5-0.9 final volume of 4.0mL with TE traditionally been accomplished in in LB broth with chloramphenicol buffer swinging bucket rotors spinning for (0.2µg/mL) • Add 400µL ethidium bromide (EtBr) 72 to 96 hours and often required • Harvest cells in a 250mL tissue cul- (10mg/mL) and 4.4g of solid cesium large volumes of cell material (Anet ture tube by centrifuging in a chloride (CsCl). The refractive index and Strayer, 1969). However, with Thermo Scientific Sorvall RC-3B after mixing is n = 1.3865 the introduction of vertical and fixed Plus centrifuge at 2,119 x g (2,861 (1.35g/mL). angle rotors, the centrifugation times rpm) for 10 minutes at 20°C with • Load solution into Thermo Scientific and starting material amounts have the H-6000A swinging bucket rotor UltracrimpTM tube, overlay with min- been significantly reduced leading to • Lyse cells by boiling method using eral oil and seal • Spin tube overnight in a Stepsaver laboratory productivity is increased 65V13 rotor at 192,553 x g while the life of the rotors and in- (45,000 rpm) at 20°C in a strument are extended. Thermo Scientific ultraspeed centrifuge Isolation and Purification of Plasmid • Visualize DNA bands under long- DNA from Samples Using the Thermo Scientific TFT80.2 Fixed Angle wave UV light chromosomal DNA Ultraspeed Centrifuge Rotor Robertc Martin and William Schneider Step Run Microbiology Division • Prepare samples and load into plasmid DNA Michigan Department of Public Ultracrimp centrifuge tubes as Health Laboratories, Lansing MI described for standard protocol • Centrifuge tubes in Stepsaver A strain of bacteria causing a community or nosocomial (hospi- 65V13 rotor at 308,941 x g Figure 1: Standard protocol run overnight at 192, (57,000 rpm) for 3 hours followed 553 x g (45,000 rpm). tal-associated) outbreak of disease by centrifugation at 192,553 x g can be “fingerprinted” using plas- (45,000 rpm) for 3 hours at 20°C mid content. DNA purification using CsCl gradients can allow for Layered Sample the identification and comparison • Prepare CsCl cushion by mixing of similar but structurally different 3.5mL TE buffer, 360µL EB plasmids from isolates of suspected (10mg/mL) and 4.4g CsCl. strains. Such gradients, although • Add solution to Ultracrimp tube useful, are usually not practical due to the large volumes of cell material • Add 40µL EB (10mg/mL) to chromosomal DNA 500uL of DNA sample that often must be used to visualize • Gently layer DNA/EtBr sample bands, and due to the fact that plasmid DNA on top of the CsCl cushion in the some medically important bacteria Neisseria gonorrhoeae, Le- Ultracrimp tube, overlay with (e.g., gionella, spp.) mineral oil and seal do not grow rapidly • Centrifuge tube in Stepsaver Figure 2: Step-run, 3 hours at 308,941 x g or in broth culture. 65V13 rotor at 308,941 x g (57,000 rpm) followed by 3 hours at 192,553 x g In this application brief, the lab- (45,000 rpm). (57,000 rpm) for 3 hours at 20°C oratory of Martin and Schneider has found the method of Kado and Results Liu, with slight modifications using The banding pattern for chromoso- the fixed angle TFT80.2 ultraspeed mal (top band) and plasmid (bot- centrifuge rotor, to be the simplest tom band) DNA in a typical technique and the best technique overnight run at 192,553 x g for minimizing contamination of plasmid preparations with chromo- c (45,000 rpm) in the Stepsaver 65V13 vertical rotor is shown in chromosomal DNA somal debris from small volumes of Figure 1. The bands are well plasmid DNA bacterial cells. spaced, ensuring easy DNA re- Procedure moval, yet tight enough to allow DNA Preparation recovery of the DNA in the minimal amount of volume. The banding • Extract DNA by addition of phe- pattern obtained for a 6 hour step- nol-chloroform Figure 3: Layered sample three hours at • Precipitate DNA by addition of run (Figure 2, 3 hours at 308,941 x 308,941 x g (57,000 rpm). g (57,000 rpm) followed by 3 hours ammonium acetate and cold 2- propanol for 30 minutes at -20°C at 192,553 x g (45,000 rpm) is sim- Discussion ilar to that seen for the single • Pellet DNA in microcentrifuge for Using the Thermo Scientific Step- 30 minutes at 4°C overnight spin. Figure 3 illustrates saver 65V13 and the ultracentrifuge the results seen when using the lay- • Carefully decant propanol and re- step-run feature, altering the prepa- move remaining propanol by ered sample technique. A banding ration of the sample and using lay- pattern similar to that seen in the evaporation under nitrogen ered sample technique, plasmid • Rehydrate pellet in 1mL TE typical overnight run was obtained isolations on CsCl gradients can be in only three hours, spinning at buffer (10mM Tris, 1mM EDTA, completed within a convenient work- pH 8.0) 308,941 x g (57,000 rpm). ing day. By using these techniques, Method for Density Gradient University of Texas, Health Science and transfer the upper aqueous Centrifugation Center, San Antonio, Texas 78284 phase to a new tube and repeat • For CsCl purification, add 1mL of Using our fixed angle TFT 80.4 ul- • Transfer the upper aqueous phase plasmid preparation to 2mL traspeed rotor and the procedure to a new tube and extract with an polypropylene ultraspeed cen- described below, we were able to equal volume of chloroform trifuge tube isolate several plasmids carrying • Separate phases by centrifugation • Add 1g of CsCl (approx. density human Factor IX and X gene frag- at 20 - 30,000 x g for 2 minutes 1.6g/mL) ments in a small volume of 3.5 mL • Transfer the upper aqueous phase • Cover with Parafilm M® and gen- in less than three hours. into a new tube and add 1 volume tly invert several times until CsCl of 3M sodium acetate (pH 7.0) goes into solution Procedure followed by 2 volumes of ethanol. • Add 80µL EtBr (10mg/mL) and Keep on dry ice for 20 minutes place cap on tube • Centrifuge at 20 - 30,000 x g for • Fill remaining space in tube with TE 15 minutes buffer using a needle and syringe • Decant the supernatant and wash • Put set-screw into place with 70% alcohol and dry the • Centrifuge material in TFT80.2 Procedure pellet in a speed vac rotor for 16 hours (or overnight) • Dissolve the pellet in 510 µl TE at 292,330 x g (60,000rpm) (10 mM Tris HCl, 1 mM EDTA, • Visualize plasmid DNA under pH 8.0) buffer in a microtube UV light • Collect DNA by puncturing side Method for Density Gradient of tube with a 20 gauge needle Centrifugation and slowly withdraw lower of • Add 900 mg CsCl to DNA solu- tion and vortex to dissolve salt two resulting bands DNA Preparation • Spin the sample at 12,000 x g for • Remove ethidum (Et) by washing • Pellet bacterial cells from 10 minutes to float any (x5) with isopropanol and desalt overnight culture by centrifuga- pellet which forms tion at > 6,000 x g in the SLA- • Transfer the liquid from below Discussion 1500 for 10 minutes at 4°C and the protein pellet to another mi- Multiple strains of Neisseria gonor- discard the supernatant crotube and add 100 µL EtBr rhoeae are prevalent in this country. • Resuspend the cell pellet in 2 mL (10 mg/mL) This procedure allowed us to iden- of buffer (50 mM glucose, 10 • To a 3.5 mL Ultracrimp™ tube add tify the strain of penicillinase-pro- mM EDTA, 25 mM Tris, pH 8.0) 1.6 mL CsCl (prepared by adding ducing N. gonorrhoeae epidemic in and transfer to a 35 mL 0.65 g CsCl to 1 mL TE buffer) southeastern Michigan as a strain polypropylene tube • Underlay the CsCl with the possessing a 3.4MD betalactamase • Add 0.5 mL of lysozyme, (20 DNA/EtBr/CsCl sample producing plasmid instead of the mg/mL in H O) and mix thor- 2 • Gently fill the remaining portion 4.7MD plasmid identified in pre- oughly. Incubate on ice for 10 of the tube with CsCl solution vous Michigan isolates. We have minutes and seal also used this technique to recover • Add 5 mL of 0.2M NaOH, 1% • Spin for 2.5 hours at 359,500 x g a large quantity of a 72 MD plas- SDS and mix gently. Incubate on (70,000 rpm) at 20°C in the TFT mid associated with fimbriae pro- ice for 10 minutes 80.4 rotor duction in strains of Escherichia • Add 4 mL of potassium acetate • Visualize the DNA bands under coli 0157:H7, an organism a- (29.4g potassium acetate; 11.5 UV light ssociated with hemorrhagic colitis. mL glacial acetic acid. Add H O 2 • Slice off the top of the tube and The TFT80.2 ultraspeed cen- to 100 mL) and mix thoroughly). draw material off the top of the trifuge rotor significantly simplifies Incubate on ice for 10 minutes. tube with a Pasteur pipet the recovery of purified covalently • Spin down the precipitate by cen- • Discard all of the material above closed circular forms of both small trifugation at 20 - 30,000 x g in the plasmid, and save the lower and large plasmid DNA from small an SS-34 for 15 minutes at 4°C plasmid band. volumes of bacterial cells. Using and transfer the supernatant to a • Extract the EtBr twice with equal this we are able to identify specific new tube volumes of isopropyl alcohol (sat- pathogenic bacterial strains which • Add heat-inactivated RNAse A urated with CsCl) will enable us to more clearly evalu- (20 µg/mL) to supernatant and in- • Add 9 volumes of 0.3 M sodium ate treatment and control measures. cubate for 15 minutes at room acetate to the aqueous DNA sam- temperature Rapid Isolation of Small Volume ple followed by 20 volumes of • Extract with an equal volume of Plasmid DNA Using TFT80.4 Rotor cold ethanol. Keep on dry ice for H O-saturated phenol:chloro- Dr. Pudur Jagadeeswaran and Dr. 2 20 minutes and spin for 20 min- form (1:1) Kavala Jayantha Rao utes at 20 - 30,000 x g • Separate phases by centrifugation Department of Cellular and Struc- • Discard the supernatant and wash at 20 - 30,000 x g for 10 minutes tural Biology the pellet with 70% alcohol and dry it. Dissolve the pellet in a ers (Table 1). These widely avail- A Laboratory Manual. 488. Cold In addition to these offices, known quantity of TE and test able kits use a column format with Spring Harbor Laboratory. USA: Thermo Fisher Scientific the concentrations of DNA by binding matrix to bind DNA in- 86-96. maintains a network of agarose gel electrophoresis and/or stead of subjecting DNA to a CsCl representative organizations UV spectrophotometer gradient. Also, instead of ultraspeed Meselson, M., Stahl, F.W. and Vino- throughout the world. centrifugation, the procedures can be grad, J. 1957. Proc. Nat. Acad. Sci. Discussion conducted using Thermo Scientific USA. 43: 581-583. The banding pattern seen for our microcentrifuge and lowspeed experiments is typical of CsCl benchtop centrifuge models which Flamm, N.F., Bond, H.E. and Burr, banded nucleic acids. Our plasmids can decrease DNA preparation time H.E. 1966. Biochim, Biophys. Acta. were shown to be pure by elec- to under an hour. Although, these 129: 310 – 317. trophoresis. The yield of super- modern techniques are less time North America: USA / Canada coiled DNA was 1-2 mg from 1,000 consuming, some procedures may Anet, R. and Strayer, D.R. 1969. +1 800 553 0039 mL of culture broth. still require the use of the tradi- Biochem. Biophys. Res. Comm. Europe: The above protocol describes a tional methods of plasmid DNA 34: 328. Austria +43 1 801 40 0 rapid and convenient method for isolation, including the separation Belgium isolating plasmid DNA from small of different DNA isotypes (ie, heavy Kado, C.I., and S.T. Liu. 1981. +32 2 482 30 30 32 cell cultures. Using the small vol- (P ) and light DNA). Rapid procedure for detection and France ume TFT 80.4 ultraspeed rotor, the isolation of large and small plasmids. +33 2 2803 2000 procedure can be carried out in References J. Bacteriol. 145:1365-1373. Germany national toll free 08001-536 376 under three hours in a floor model Radloff, R., Bauer, W. and Vinograd, Germany international ultraspeed centrifuge, with the pu- J. 1967. Proc. Nat. Acad. Sci.USA Heilig, J.S., Elbing, Karen L., and +49 6184 90 6940 rity and yield typical of larger vol- 57: 1514-1521. Roger Brent. 1998. Current Proto- Italy ume rotors. cols in Molecular Biology. John +39 02 02 95059 341 Maniatis, T., Fritsch, E.F. and Sam- Wiley & Sons, Inc: 1.7.1-1.7.16 Netherlands +31 76 571 4440 Conclusion brock, J. 1982. . Nordic countries A variety of techniques have been +358 9 329 100 established over the years to isolate Russia / CIS plasmid DNA from crude lysate, in- +7 (812) 703 42 15 cluding alkaline, boiling, and Triton COMPANY PRODUCT LINE PURIFICATION BINDING MATRIX CAPABILITIES Spain / Portugal FORMATION +34 93 223 09 18 mediated (Heilig et al., 1998). Bio-Rad Labora- Quantum Prep Spin Diatomaceous Mini, midi-, and Switzerland However, density gradient centrifu- tories, Inc.. Plasmid Purifica- earth maxi-prep +41 44 454 12 12 gation as described in this applica- tion Kits UK / Ireland tions brief is the method of choice Bio-Rad Labora- Aurum Mini Kit Spin or vacuum Silica membrane Mini-prep +44 870 609 9203 amongst the traditional techniques tories, Inc. Asia: China ® because it yields high-quality plas- Clontech Nucleobond Spin or vacuum Nucleobond AX Mini-, midi-, and +86 21 6865 4588 or mid DNA free of most contami- Kits (silica based maxi-prep +86 10 8419 3588 nants (Heilig et al., 1998). The anion resin ex- India change) biggest drawbacks with CsCl/EtBr +91 22 6716 2200 Clontech NucleoSpin® Spin or vacuum Silica membrane 96 well, mini- centrifugation are the long spin Japan midi-, and maxi- +81 45 453 9220 times and the use of ethidium bro- prep Other Asian countries mide (a mutagen). Mo Bio Labora- Ultra Clean™ Spin Silica membrane Endotoxin-free +852 2885 4613 tories, Inc. Kits 96 well, mini-. Countries not listed: Alternative Methods to the midi-, maxi-prep +49 6184 90 6940 or +33 2 2803 2000 Traditional Approach QIAGEN QIAprep Spin Spin or vacuum Silica gel 96 well, mini- www.thermo.com/centrifuge Recently, less time consuming and membrane prep less hazardous methods for the iso- QIAGEN QIAGEMN Spin Anion-exchange Endotoxin-free © 2008 Thermo Fisher Scientific Plasmid Kits and resin mini-, midi-, Inc. Nucleobond and NucleoSpin lation of plasmid DNA from small are registered trademarks of QIAFilter Kits maxi-, mega-prep and large scale volumes have been Clontech. Parafilm M is a regis- Sigma-Aldrich, GenElute™ Spin or vacuum Silica membrane Endotoxin-free tered trademark of life American National Can Company. Ultra developed. Commercial plasmid Inc. 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