ChaparI Petat aunt of smd DNA by Saal-sat Bodin Lyss Cold Spring harbor Molecular Cloning tory Prti A LABORATORY MANUAL ON THE WEB CHAPTER 1>PROTOCOL 4 0 printer friendly version Protocol 4 Preparation of Plasmid DNA by Small-scale Boiling Lysis asmid DNA is isolated from small-scale( 1-2 ml) bacterial cutures by treatment with Tnton X-100 and lysozyme followed by heating. This method is not recommended for preparing plasmid DNA fom strains of E coli fhat express endonuclease A(endA" strains) MATERIALS A CAUTION: Please dick for information about appropriate handling of materials O RECIPE: Please dick for components of stock solutions, buffers, and reagents Buffers and solutions Antibiotic for plasmid selection Ethand Isopropanol 0 Sodium acetate(3.0W pH 52 O TE (pH8. 0)containing 20 ulmi RNase A zymes and Buffers 0 Lysozyme(10 mgiml) O Rich medium METHOD L. hoculate 2 m of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria Incubate the cuture ovemight at 37 C with vigorous shaking 2. Pour 1.5 ml of the culture into a miarofuge tbe Centrifuge the tibe at maximum speed for 30 seconds at 4 C in a microfuge. Store the unused portion of the cuture at 4'C 3. Remove the medium by gentle aspiration, leaving the bacterial pellet as dry as poss ble 4. Resuspend the bacterial pellet in 350 pl ofSTE ). Add 25 p of a freshly prepared soluton of lysozyme. Close the top of the tube and mix the contents by gently vortexing 6. Place the tube in a boing water bath for exactly 40 seconds 7. Centrifuge the bacterial lysate a maximum speed for 15 minutes at room temperature in a microfuge. Paur the supematant in o a fresh miarofuge tube 8. Precipitate the nucleic acids from the supematant by adding 40 pl of 2.5 W sodium acetate(pH 5.2) and 420 pl of propanol. Mix the solution by vortexing, and then allow the mixture to stand for 5 minutes at room temperature Recover the precipitated nucleic acas by centrifugation at maximum speed for 10 minutes at 4'C in a microfuge 10. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow al of the fluid to drain away. Use a Kimwipe or disposable pipette ip to remove any drops of fud adhering to the was of the tube. IL. Rinse the pellet of nudeic acid with 1 ml of 70% ethanol at 4'C. Remove all of the supematant by gente aspiration as described n Step 3. 12. Remove any beads of ethanol that form on the sides of the tube Store the open tube at room temperature urti the ethanol has evaporated and o fluid is visible in the tube ( 2-5 minutes) 13. Dissolve the nucleic acids in 50 ul of TE (pH 8.0)containing DNase-free RNase A (pancreatic RNase). Vortex the solution genty for a bnef period Store the DNA al-20C REFERENCES L. Holmes D.S. and Quigley M, 1981. A rapid boiling method for the preparafion of bacterial plasmids. Anal. Biochem. 114193197 o printer friendly version Bu e BookI our Vein Late The Tou Newslette I Search CSi Press Home I Cantact Members Home ICSHHome Copyright92000 Cad Spring Harbor Laboratory Press. htp hwwnoeaarnigcommembersproblipspprorumber4achenumbera1 [2002-2-18 1811:31Chapter:1 Protocol:4 Preparation of Plasmid DNA by Small-scale Boiling Lysis CHAPTER 1 > PROTOCOL 4 printer friendly version Protocol 4 Preparation of Plasmid DNA by Small-scale Boiling Lysis Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with Triton X-100 and lysozyme, followed by heating. This method is not recommended for preparing plasmid DNA from strains of E. coli that express endonuclease A (endA+ strains). MATERIALS CAUTION: Please click for information about appropriate handling of materials. RECIPE: Please click for components of stock solutions, buffers, and reagents. Buffers and Solutions Antibiotic for plasmid selection Ethanol Isopropanol Sodium acetate (3.0 M, pH 5.2) STET TE (pH 8.0) containing 20 µg/ml RNase A Enzymes and Buffers Lysozyme (10 mg/ml) Media Rich medium METHOD 1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking. 2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge the tube at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the culture at 4°C. 3. Remove the medium by gentle aspiration, leaving the bacterial pellet as dry as possible. 4. Resuspend the bacterial pellet in 350 µl of STET. 5. Add 25 µl of a freshly prepared solution of lysozyme. Close the top of the tube and mix the contents by gently vortexing for 3 seconds. 6. Place the tube in a boiling water bath for exactly 40 seconds. 7. Centrifuge the bacterial lysate at maximum speed for 15 minutes at room temperature in a microfuge. Pour the supernatant into a fresh microfuge tube. 8. Precipitate the nucleic acids from the supernatant by adding 40 µl of 2.5 M sodium acetate (pH 5.2) and 420 µl of isopropanol. Mix the solution by vortexing, and then allow the mixture to stand for 5 minutes at room temperature. 9. Recover the precipitated nucleic acids by centrifugation at maximum speed for 10 minutes at 4°C in a microfuge. 10. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube. 11. Rinse the pellet of nucleic acid with 1 ml of 70% ethanol at 4°C. Remove all of the supernatant by gentle aspiration as described in Step 3. 12. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (2-5 minutes). 13. Dissolve the nucleic acids in 50 µl of TE (pH 8.0) containing DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a brief period. Store the DNA at -20°C. REFERENCES 1. Holmes D.S. and Quigley M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193-197. printer friendly version Buy The Book | Our Vision | Take The Tour | Newsletter | Search CSHL Press Home | Contact | Members Home | CSHL Home Copyright © 2000. Cold Spring Harbor Laboratory Press. http://www.molecularcloning.com/members/protocol.jsp?pronumber=4&chpnumber=1 [2002-2-18 16:11:35]