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Traditional Methods for Cscl Isolation of Plasmid DNA by Ultracentrifugation

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Traditional Methods for Cscl Isolation of Plasmid DNA by Ultracentrifugation Application Note: AN-LECF- Traditional Methods for CsCl Isolation PLASDNA-0408 of Plasmid 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 Plasmids are pieces of extra-chromo- instrumentation and rotor life. lysozyme • Density Gradient somal DNA found in bacteria. 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 ethanol 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.
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