Chaper. I PetaatI Aepuntm of md DNA by Alaine Lyss wth SD: Minpmepardie Cold Spring harbor Molecular Cloning tory Prti A LABORATORY MANUAL ON THE WEB CHAPTER 1>PROTOCOL 1 0 printer friendly version Protocol 1 Preparation of Plasmid DNa by Alkaline Lysis with SDS: Minipreparation Plasmid DNA s isdated from smal-scale (1-2 ml) bacterial cutures by treatment with alkali 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 0 Alkaine lysis solufion ll 0 Alkaline lysis solufion I Antibiotic for plasmid selection △ Phenolchloroform O STE O TE (pH 8.0) containing 20 uglml RNase A 0 Rich METHOD I. hoculate 2 m of rich medium(LB, YT, or Terific 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 at maximum speed for 30 seconds at 4 C in a microfuge Store the unused portion of the original ature at4C 3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible 4. Resuspend the bacterial pellet in 100 ul of ice-cold Akaline lysis solution I by vigorous vortexing. 5. Add 200 ul of freshly prepared Alkaline lysis soluton l to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Donat vortex/ Store the tube on ice 6. Add 150 ul of ice-cold Alkaline lysis solution l. Close the tube and disperse Akaline lysis solufion ll through the vicous 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 the supematant to a fresh tube 8. (optional Add an equa volume of phenol chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed far 2 minues at 4 C ina microfuge. Transfer the aqueous upper layer to a 9. Precipita e nuleic adids from the supematant by adding 2 volumes of ethanol at room temperature. Mix the solution by ortexing and then aow the mixture to stand for 2 minutes at room temperature 10. Called the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4 C in a microfuge IL. Remove the supernatant by gentle aspiration as described n Step 3 above. Stand the tube in an inverted position on a paper towel to allow al of the flud to drain away. Use a Kmwipe ar disposable pipette tip to remove any drops of fud adhering to the wals of the tube. 12. Add 1 ml of 70% ethanol to the pellet and invent the dosed t be several times. Recover the DNA by certrifugation at maximum speed for 2 minutes at 4 C in a microfuge 13. Remove all of the supematant by gentle aspiration as described in Step 3. Take care with this step, as the pellet sometimes does not adhere tightly to the tube I4. 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 mo flud is visible in the tube( 5-10 minutes) 15. Dissolve the nucleic acids in 50 ul of TE (pH 8.0) containing 20 ug ml DNase-free RNase A(panceatic RNase) Vortex the solution gently for a few seconds. Store the DNA solution at-20'C REFERENCES L. Bimboim H C and Doly J. 1979. A rapid alkaine procedure for screening recombinant plasmid DNA. Nucleic Acids Re7:1513-1523 2. sh-Horowicz D. and Burke JF. 1981. Rapid and effcient cosmid doning. Nucleic Acids Res. 9.2989-2998 0 printer friendly version Buy The Book I Our Vision I Take The Tour I Newsletter I Search CSH. Press Home CAnad embers Home ICSH. Home htp hwwnoeaarnigcommembersproblipspprorumbertachenumbera1 [2002-2-18 16211: 01Chapter:1 Protocol:1 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation CHAPTER 1 > PROTOCOL 1 printer friendly version Protocol 1 Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation Plasmid DNA is isolated from small-scale (1-2 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 Alkaline lysis solution II Alkaline lysis solution III Antibiotic for plasmid selection Ethanol Phenol:chloroform (1:1, v/v) STE TE (pH 8.0) containing 20 µg/ml RNase A 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 at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C. 3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible. 4. Resuspend the bacterial pellet in 100 µl of ice-cold Alkaline lysis solution I by vigorous vortexing. 5. Add 200 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 150 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 the supernatant to a fresh tube. 8. (Optional) 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 2 volumes of ethanol 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 4°C 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. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube. 12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge. 13. Remove all of the supernatant by gentle aspiration as described in Step 3.Take care with this step, as the pellet sometimes does not adhere tightly to the tube. 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 (5-10 minutes). 15. Dissolve the nucleic acids in 50 µl of TE (pH 8.0) containing 20 µg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds. Store the DNA solution 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=1&chpnumber=1 [2002-2-18 16:11:00]