CaparI Petats hepuntm of md DNAT中Mn中m Cold Spring harbor Molecular Cloning tory Prti A LABORATORY MANUAL ON THE WEB CHAPTER 1> PROTOCOL 6 0 printer friendly version Protocol 6 Preparation of Plasmid DNA: Toothpick Minipreparation Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar media with toothpicks. 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 Antibiotic for plasmid selection O Bromophenol blue solution(0.4%ww 0 Cresol red solution(10 mM) O EDTA(0.5M, PH 8O) AO Ethidium bromide(10 mgm) AOSYBR Gold 0 KCI(4M) 0 NSS solution 0 Rich broth 0 Rich broth agar plates Additional Reagents Step 12 of this protocol requires the reagents listed in Chapter 8 Protocd 1. Step 14 of this protocol requires the reagents listed in Chapter 1, Protocd 1 or Chapter 1, Protocol 4 METHOD L. Grow bacterial colonies, transformed with recombinant plasmid, on rich agar medium(LB, YT, or SOB)containing the appropriate antibioticunfil they are approx. 2-3 mm in diameter(approx 18-24 hours at 37 C for most bacterial strains) 2. Use a sterile toothpick or disposable loop to transfer a small segment of a bacterial colomy to a streak or patch on a master agar plate containing the appropriate antibiotic. Transfer the remainder of the colony to a numbered microfuge containing 50 ul of sterile 10 mM EDTA(PH 8.0). 4. haubate the master plate for several hours at 37 C and then store it at 4 C until the results of the gel electrophoresis Step 11 of this protocol) are available. Colonies cantaining plasmids of the desired size can then be recovered from the master plate 5. While the master plate is incubating process the bacterial suspensions as follows: To each microfuge tube in tum, add 50 pl of a freshly made solution of NSS. Close the top of the tubes and then ma their contents by vortexing for 30 seconds 6. Transfer the tubes to a 70'C water bath. Incubate the tubes for 5 minutes and then alow them to cool to room 1. To each the, ad 1.5 p of a solution of 4 M KCl Vortex the tubes for 30 seconds 8. incubate the tutes for 5 minutes n ice 9. Remove bacterial debris by centrifugation at maximum speed for 3 minutes at 4 C in a microfuge 10. Transfer each of the supematants in turn to fresh microfuge ttes. Add to each tube 0.5 pl of a solufion containing 0.4% bromophenol blue if the samples are to be analyzed only by agarose gel electrophoresis or 2 ul of 10 mM cresol red if the samples are to be analyzed both by pCr and by agarose gel electrophoresis. Load 50 p of the supernatant into a slot (5 mmin length x 2.5 mm in width) cast in a 0.7% agarose gel (5 mm thick Il. After the bromophenol blue dye has migrated two-thirds to three-fourths the length of the gel, or the cresol red dye about one- haf the length of the gel, stain the gel by soaking it for 30-45 minutes in a DNA-staining solution at room temperature. Examine and photograph the gel under UV ilumination. I2 Maes red has been used at Step 10, analyze the supematants by performing PCR as described in Chapter 8 Protocol 1, using the remainder of each sample as a template 13. Prepare smal-scale cultures of the putative recombinant clones by inocula ig 2 ml of iquid medium( LB, YT, or SOB) containing the appropriate antibiotic with bactenia growing on the master plate I4. Use the smal-scale bacterial cutures to generate minipreparations(please see Chapter 1, Protocol 1 or Chapter 1. Protocol 4)of the putative recombinant plasmids. Analyze the plasmid DNAs by digestion with restriction enzymes and agarose gel electrophoresis to confirm that they have the desired size and structure REFERENCES Bames W.M. 1977 Plasmid detedion and sizing in single colomy lysates. Science 195.393-394 0 printer friendly version Book I ur Visin I Tate The Tour Newslette I Search Press Home ICants I Members Home ICSH Home Copyright 82000. Cold Spring Harbor Laboratory Press. htp hwwnoeaarnigcommembersproblipspprorumber6chenumbera1 [2002-2-18 18211: 53)Chapter:1 Protocol:6 Preparation of Plasmid DNA: Toothpick Minipreparation CHAPTER 1 > PROTOCOL 6 printer friendly version Protocol 6 Preparation of Plasmid DNA: Toothpick Minipreparation Plasmid DNA is prepared directly from bacterial colonies plucked from the surface of agar media with toothpicks. 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 Bromophenol blue solution (0.4% w/v) Cresol red solution (10 mM) EDTA (0.5 M, pH 8.0) Ethidium bromide (10 mg/ml) SYBR Gold KCl (4 M) NSS solution Media Rich broth Rich broth agar plates Additional Reagents Step 12 of this protocol requires the reagents listed in Chapter 8, Protocol 1 . Step 14 of this protocol requires the reagents listed in Chapter 1, Protocol 1 or Chapter 1, Protocol 4 . METHOD 1. Grow bacterial colonies, transformed with recombinant plasmid, on rich agar medium (LB, YT, or SOB) containing the appropriate antibiotic until they are approx. 2-3 mm in diameter (approx. 18-24 hours at 37°C for most bacterial strains). 2. Use a sterile toothpick or disposable loop to transfer a small segment of a bacterial colony to a streak or patch on a master agar plate containing the appropriate antibiotic. Transfer the remainder of the colony to a numbered microfuge tube containing 50 µl of sterile 10 mM EDTA (pH 8.0). 3. Repeat Step 2 until the desired number of colonies has been harvested. 4. Incubate the master plate for several hours at 37°C and then store it at 4°C until the results of the gel electrophoresis (Step 11 of this protocol) are available. Colonies containing plasmids of the desired size can then be recovered from the master plate. 5. While the master plate is incubating, process the bacterial suspensions as follows: To each microfuge tube in turn, add 50 µl of a freshly made solution of NSS. Close the top of the tubes and then mix their contents by vortexing for 30 seconds. 6. Transfer the tubes to a 70°C water bath. Incubate the tubes for 5 minutes and then allow them to cool to room temperature. 7. To each tube, add 1.5 µl of a solution of 4 M KCl. Vortex the tubes for 30 seconds. 8. Incubate the tubes for 5 minutes on ice. 9. Remove bacterial debris by centrifugation at maximum speed for 3 minutes at 4°C in a microfuge. 10. Transfer each of the supernatants in turn to fresh microfuge tubes. Add to each tube 0.5 µl of a solution containing 0.4% bromophenol blue if the samples are to be analyzed only by agarose gel electrophoresis or 2 µl of 10 mM cresol red if the samples are to be analyzed both by PCR and by agarose gel electrophoresis. Load 50 µl of the supernatant into a slot (5 mm in length x 2.5 mm in width) cast in a 0.7% agarose gel (5 mm thick). 11. After the bromophenol blue dye has migrated two-thirds to three-fourths the length of the gel, or the cresol red dye about one-half the length of the gel, stain the gel by soaking it for 30-45 minutes in a DNA-staining solution at room temperature. Examine and photograph the gel under UV illumination. 12. If cresol red has been used at Step 10, analyze the supernatants by performing PCR as described in Chapter 8, Protocol 1 , using the remainder of each sample as a template. 13. Prepare small-scale cultures of the putative recombinant clones by inoculating 2 ml of liquid medium (LB, YT, or SOB) containing the appropriate antibiotic with bacteria growing on the master plate. 14. Use the small-scale bacterial cultures to generate minipreparations (please see Chapter 1, Protocol 1 or Chapter 1, Protocol 4 ) of the putative recombinant plasmids. Analyze the plasmid DNAs by digestion with restriction enzymes and agarose gel electrophoresis to confirm that they have the desired size and structure. REFERENCES 1. Barnes W.M. 1977. Plasmid detection and sizing in single colony lysates. Science 195:393-394. 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=6&chpnumber=1 [2002-2-18 16:11:58]