Chaper. I Peta2 Aepuntom of md DNA by Alaine Lyss wth SD: Mbdpmepardie Cold Spring harbor Molecular Cloning tory Prti A LABORATORY MANUAL ON THE WEB CHAPTER 1>PROTOCOL 2 0 printer friendly version Protocol 2 Preparation of Plasmid DNa by Alkaline Lysis with SDS: Midipreparatiot Plasmid DNA is isdated from intermediate-scale(20-50 m) bactenial cultures by treatment with a kal and SDS MATERIALS A CAUTON: Please dick for information about appropriate handing of materias. O RECIPE: Please dik for components of stock solutions, buffers, and reagents Buffers and solutions O Alkaine lysis solufion I Far preparations of plasmid DNA that are to be subjected to further pumica ion by chromatography (please see Chapter 1, Protocol 9), sterile Alkaline lais solution I may be supplemented just before use with the appmpriat volume df 20 mgm/ DNase-free RNase A / pancreatic RNase)to give a final concentration of 100 ug/ml 0 Alkaline lysis solufion ll 0 Alkaline lysis solution Il biotic for plasmid selection △ Phenotchoroform O STE O TE (pH 8. 0)containing 20 ugliml RNase A 0 Rich medium METHOD I. inoculate 10 ml of rich medium(LB, YT, or Temfic Broth) containing the appropriate antibiotic with a single colony of transformed bactera Incubate the cuture ovemight at 37 wih vigorous shaking 2. Transfer the ature into a 15-ml tube and recover the bacteria by centrifugation at 200g(400 pm in a Sorvall SS-34 rtn1mnu爬a4 3. Remove the medium by gentle aspiration, leaving the bacterial pellet as dry as possible 4. Resuspend the bacterial pellet in 200 ul of ice- dld Akaline lysis solution I by vigorous vortexing and transfer the suspension to a microfuge tube 5. Add 400 ul of freshly prepared Alkaline lysis soluton Il to each bacterial suspension. Close the tube tightly, and mix the ontents by inverting the tube rapidly five fimes. Do not vortex/ Stare the tube on ice 6. Add 300 pl of ice-cold Alka ne lysis solution ll. Close the tube and disperse Akaline lysis solufion ll through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes 1. Centrifuge the bacterial lysate at maximum speed for 5minutes at 4"C in a miarofuge. Transfer 600 p of the supematant to a fresh tube 8. Add an equal volume of pheno chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4 c ina miarofuge Transfer the aqueous upper layer to a fresh tbe 9. Precipitate nudeic acids from the supematant by adding 600 pl of isopropanol at room temperature. Mix the solution by artexing and then allow the mixture to stand for 2 minutes at room temperature 10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at room temperature in a microfuge 々 sper towel to allow al of thet如em时h动mgt能的m Remove the supematant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted posit d1 ml of 70% ethanol to the pellet and recover the DNA by centrifugafion af maximum speed for 2 minutes at room temperature in a microfuge 13. Remove all of the supematant by gentle aspiration as desaibed in Step 3 I4. Remove any beads of ethanol that form on the sides of the tube Store the open tube at room temperature unti the ethano has evaporated and no fluid is visible in he tube ( 2-5 minutes) 15. Dissolve the nucleic acids in 100 ul of TE (pH 8. 0) containing 20 ugml DNase-fee RNase A(pancreatic RNase) Vortex the solution gently for a few seconds and store at -20C REFERENCES L. Bimboim H.C. and Doly J. 1979. A rapid alkaline procedure for screening recombinant plasmid DNA Nucleic Acids Res7:15131523 2. ksh-Horowicz D and Burke JF. 1981. Rapid and efficient cosmid doning. Nucleic Acids Res. 9.2989-2998 0 printer friendly version ByTe BookI Qur Vsn Taie The Tour Newsletter I Searh CSH. Press Home cOmtac Members Home CSHLHme ygt92000 Cdd Spring Harbor Laboratory Press htp hwwnoeaarnigcommembersproblipspprorumber chenumbera1 [2002-2-18 1E11:10)Chapter:1 Protocol:2 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation CHAPTER 1 > PROTOCOL 2 printer friendly version Protocol 2 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Midipreparation Plasmid DNA is isolated from intermediate-scale (20-50 ml) bacterial cultures by treatment with alkali and SDS. 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 Alkaline lysis solution I For preparations of plasmid DNA that are to be subjected to further purification by chromatography (please see Chapter 1, Protocol 9 ), sterile Alkaline lysis solution I may be supplemented just before use with the appropriate volume of 20 mg/ml DNase-free RNase A (pancreatic RNase) to give a final concentration of 100 µg/ml. Alkaline lysis solution II Alkaline lysis solution III Antibiotic for plasmid selection Ethanol Isopropanol Phenol:chloroform (1:1, v/v) STE TE (pH 8.0) containing 20 µg/ml RNase A Media Rich medium METHOD 1. Inoculate 10 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. Transfer the culture into a 15-ml tube and recover the bacteria by centrifugation at 2000g (4000 rpm in a Sorvall SS-34 rotor) for 10 minutes at 4°C. 3. Remove the medium by gentle aspiration, leaving the bacterial pellet as dry as possible. 4. Resuspend the bacterial pellet in 200 µl of ice-cold Alkaline lysis solution I by vigorous vortexing, and transfer the suspension to a microfuge tube. 5. Add 400 µl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice. 6. Add 300 µl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes. 7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microfuge. Transfer 600 µl of the supernatant to a fresh tube. 8. Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube. 9. Precipitate nucleic acids from the supernatant by adding 600 µl of isopropanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature. 10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at room temperature in a microfuge. 11. 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. Remove any drops of fluid adhering to the walls of the tube. 12. Add 1 ml of 70% ethanol to the pellet and recover the DNA by centrifugation at maximum speed for 2 minutes at room temperature in a microfuge. 13. Remove all of the supernatant by gentle aspiration as described in Step 3. 14. 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). 15. Dissolve the nucleic acids in 100 µl of TE (pH 8.0) containing 20 µg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds and store at -20°C. REFERENCES 1. Birnboim H.C. and Doly J. 1979. A rapid alkaline procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 2. Ish-Horowicz D. and Burke J.F. 1981. Rapid and efficient cosmid cloning. Nucleic Acids Res. 9:2989-2998. 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=2&chpnumber=1 [2002-2-18 16:11:10]