@片Molecular Cloning Protocol 24 and of CompetentCoUra-CompCe Cgo0ee6aetatresesesepn soc medum METHOD tor Final c IPES M..7) ration through a prerinsed 0.45-um Nalgene flter.Divide into alquots ar 2-3 5 250.300 a12500g300pm 15.U . 16. 90-mm plate)of transformed competent cells onto agar 50B mediun er=1 (1/)2002--18 1-1
Chapter:1 Protocol:24 The Inoue Method for Preparation and Transformation of Competent <i>E. Coli</i>: "Ultra-Competent" Cells CHAPTER 1 > PROTOCOL 24 printer friendly version Protocol 24 The Inoue Method for Preparation and Transformation of Competent E. Coli: "Ultra-Competent" Cells This protocol reproducibly generates competent cultures of E. coli that yield 1 x 108 to 3 x 108 transformed colonies/mg of plasmid DNA. The protocol works optimally when the bacterial culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can be set up in a 4°C cold room and regulated to 18°C. 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 DMSO Inoue transformation buffer (please see Step 1) Chilled to 0°C before use. Nucleic Acids Plasmid DNA (recombinant plasmid) Construct using one of the methods described in Chapter 1, Protocol 17 , Chapter 1, Protocol 18 , Chapter 1, Protocol 19 , Chapter 1, Protocol 20 , Chapter 1, Protocol 21 and Chapter 1, Protocol 22 . Media SOB medium for initial growth of culture SOB agar plates containing 20 mM MgSO4 and the appropriate antibiotic SOB medium, for growth of culture to be transformed SOC medium METHOD 1. Prepare Inoue transformation buffer (chilled to 0°C before use). a. Prepare 0.5 M PIPES (pH 6.7) (piperazine-1,2-bis[2-ethanesulfonic acid]) by dissolving 15.1 g of PIPES in 80 ml of pure H2O (Milli-Q, or equivalent). Adjust the pH of the solution to 6.7 with 5 M KOH, and then add pure H2O to bring the final volume to 100 ml. Sterilize the solution by filtration through a disposable prerinsed Nalgene filter (0.45-µm pore size). Divide into aliquots and store frozen at -20°C. b. Prepare Inoue transformation buffer by dissolving all of the solutes listed below in 800 ml of pure H2O and then add 20 ml of 0.5 M PIPES (pH 6.7). Adjust the volume of the Inoue transformation buffer to 1 liter with pure H2O. Reagent Amount per liter Final concentration MnCl2•4H2O 10.88 g 55 mM CaCl2•2H2O 2.20 g 15 mM KCl 18.65 g 250 mM PIPES (0.5 M, pH 6.7) 10 ml 10 mM H2O to 1 liter c. Sterilize Inoue transformation buffer by filtration through a prerinsed 0.45-µm Nalgene filter. Divide into aliquots and store at -20°C. 2. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 25 ml of SOB medium (LB may be used instead) in a 250-ml flask. Incubate the culture for 6-8 hours at 37°C with vigorous shaking (250-300 rpm). 3. At about 6 o'clock in the evening, use this starter culture to inoculate three 1-liter flasks, each containing 250 ml of SOB. The first flask receives 10 ml of starter culture, the second receives 4 ml, and the third receives 2 ml. Incubate all three flasks overnight at 18-22°C with moderate shaking. 4. The following morning, read the OD600 of all three cultures. Continue to monitor the OD every 45 minutes. 5. When the OD600 of one of the cultures reaches 0.55, transfer the culture vessel to an ice-water bath for 10 minutes. Discard the two other cultures. 6. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C. 7. Pour off the medium and store the open centrifuge bottle on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to walls of the centrifuge bottle or trapped in its neck. 8. Resuspend the cells gently in 80 ml of ice-cold Inoue transformation buffer. 9. Harvest the cells by centrifugation at 2500g (3900 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C. 10. Pour off the medium and store the open centrifuge tube on a stack of paper towels for 2 minutes. Use a vacuum aspirator to remove any drops of remaining medium adhering to the walls of the centrifuge tube or trapped in its neck. 11. Resuspend the cells gently in 20 ml of ice-cold Inoue transformation buffer. 12. Add 1.5 ml of DMSO. Mix the bacterial suspension by swirling and then store it in ice for 10 minutes. 13. Working quickly, dispense aliquots of the suspensions into chilled, sterile microfuge tubes. Immediately snap-freeze the competent cells by immersing the tightly closed tubes in a bath of liquid nitrogen. Store the tubes at -70°C until needed. 14. When needed, remove a tube of competent cells from the -70°C freezer. Thaw the cells by holding the tube in the palm of the hand. Just as the cells thaw, transfer the tube to an ice bath. Store the cells on ice for 10 minutes. 15. Use a chilled, sterile pipette tip to transfer the competent cells to chilled, sterile 17 x 100-mm polypropylene tubes. Store the cells on ice. Include all of the appropriate positive and negative controls. 16. Add the transforming DNA (up to 25 ng per 50 µl of competent cells) in a volume not exceeding 5% of that of the competent cells. Swirl the tubes gently several times to mix their contents. Set up at least two control tubes for each transformation experiment, including a tube of competent bacteria that receives a known amount of a standard preparation of superhelical plasmid DNA and a tube of cells that receives no plasmid DNA at all. Store the tubes on ice for 30 minutes. 17. Transfer the tubes to a rack placed in a preheated 42°C circulating water bath. Store the tubes in the rack for exactly 90 seconds. Do not shake the tubes. 18. Rapidly transfer the tubes to an ice bath. Allow the cells to cool for 1-2 minutes. 19. Add 800 µl of SOC medium to each tube. Warm the cultures to 37°C in a water bath, and then transfer the tubes to a shaking incubator set at 37°C. Incubate the cultures for 45 minutes to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid. 20. Transfer the appropriate volume (up to 200 µl per 90-mm plate) of transformed competent cells onto agar SOB medium containing 20 mM MgSO4 and the appropriate antibiotic. 21. Store the plates at room temperature until the liquid has been absorbed. 22. Invert the plates and incubate them at 37°C. Transformed colonies should appear in 12-16 hours. http://www.molecularcloning.com/members/protocol.jsp?pronumber=24&chpnumber=1 (1 / 2) [2002-2-18 16:15:09]
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Chapter:1 Protocol:24 The Inoue Method for Preparation and Transformation of Competent <i>E. Coli</i>: "Ultra-Competent" Cells REFERENCES 1. Inoue H., Nojima H., and Okayama H. 1990. High efficiency transformation of Escherichia coli with plasmids. Gene 96:23-28. 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=24&chpnumber=1 (2 / 2) [2002-2-18 16:15:09]
Molecular Cloning ●printer friendly versio Protocol 25 n and Trar ntE.sing Calci id nt bacteria that yield 5x 10 to 2 CaCk-2HO (1M) er (TFB)(please soe Chagter 1.Protocol 23,Stop 1)may be used in Step B MaCl-CeCl solution.ice cold 50B medium for ETHOD 1.Pick a single ba "C with the I g in 30 ml or ins of E.coli sanderd TFB (1.)may be used insteed of Cacis with egul and nep 11 s onto agar S0B mediu REFERENCES
Chapter:1 Protocol:25 Preparation and Transformation of Competent <i>E. coli</i> Using Calcium Chloride CHAPTER 1 > PROTOCOL 25 printer friendly version Protocol 25 Preparation and Transformation of Competent E. coli Using Calcium Chloride This protocol, developed approx. 30 years ago, is used to prepare batches of competent bacteria that yield 5 x 106 to 2 x 107 transformed colonies/µg of supercoiled plasmid DNA. 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 CaCl2•2H2O (1 M) Standard transformation buffer (TFB) (please see Chapter 1, Protocol 23 , Step 1) may be used in Step 8. MgCl2-CaCl2 solution, ice cold Media SOB medium for initial growth of culture SOB agar plates containing 20 mM MgSO4 and the appropriate antibiotic SOC medium Nucleic Acids Plasmid DNA (recombinant plasmid) Construct using one of the methods described in Chapter 1, Protocol 17 , Chapter 1, Protocol 18 , Chapter 1, Protocol 19 , Chapter 1, Protocol 20 , Chapter 1, Protocol 21 and Chapter 1, Protocol 22 . METHOD 1. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 100 ml SOB medium (LB-may be used) in a 1-liter flask. Incubate the culture for 3 hours at 37°C with vigorous agitation, monitoring the growth of the culture. As a guideline, 1 OD600 of a culture of E. coli strain DH1 contains approx. 109 bacteria/ml. 2. Transfer the bacterial cells to sterile, disposable, ice-cold 50-ml polypropylene tubes. Cool the cultures to 0°C by storing the tubes on ice for 10 minutes. 3. Recover the cells by centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C. 4. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away. 5. Resuspend each pellet by swirling or gentle vortexing in 30 ml of ice-cold MgCl2-CaCl2 solution. 6. Recover the cells by centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor) for 10 minutes at 4°C. 7. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away. 8. Resuspend the pellet by swirling or gentle vortexing in 2 ml of ice-cold 0.1 M CaCl2 (or TFB) for each 50 ml of original culture. When preparing competent cells, thaw a 10-ml aliquot of the CaCl2 stock solution and dilute it to 100 ml with 90 ml of pure H2O. Sterilize the solution by filtration through a prerinsed Nalgene filter (0.45-µm pore size), and then chill it to 0°C. For many strains of E. coli, standard TFB ( Chapter 1, Protocol 23 ) may be used instead of CaCl2 with equivalent or better results. 9. At this point, either use the cells directly for transformation as described in Steps 10 through 16 below or dispense into aliquots and freeze at -70°C (please see Chapter 1, Protocol 23 , Step 12). Include all of the appropriate positive and negative controls. 10. To transform the CaCl2-treated cells directly, transfer 200 µl of each suspension of competent cells to a sterile, chilled 17 x 100-mm polypropylene tube using a chilled micropipette tip. Add DNA (no more than 50 ng in a volume of 10 µl or less) to each tube. Mix the contents of the tubes by swirling gently. Store the tubes on ice for 30 minutes. 11. Transfer the tubes to a rack placed in a preheated 42°C circulating water bath. Store the tubes in the rack for exactly 90 seconds. Do not shake the tubes. 12. Rapidly transfer the tubes to an ice bath. Allow the cells to chill for 1-2 minutes. 13. Add 800 µl of SOC medium to each tube. Incubate the cultures for 45 minutes in a water bath set at 37°C to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid. 14. Transfer the appropriate volume (up to 200 µl per 90-mm plate) of transformed competent cells onto agar SOB medium containing 20 mM MgSO4 and the appropriate antibiotic. 15. Store the plates at room temperature until the liquid has been absorbed. 16. Invert the plates and incubate at 37°C. Transformed colonies should appear in 12-16 hours. REFERENCES 1. Cohen S.N., Chang A.C.Y., and Hsu L. 1972. Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. 69:2110-2114. 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=25&chpnumber=1 [2002-2-18 16:15:18]
Molecular Cloning CHAPTER1>PROTOCOL2 Protocol 26 MATERIALS mMMg50,anhepropraeanmitc ne 50 ml of LB medium.the ice p22g2028nnoa6oncnano2xio 11. er of aliquots of ceils from the-7'C to are 1度 01-2 in the e eingpindcaiedabove.Amecon ant of 4-5 m ds with a fiel edroporafion cuvette and add 1 ml of SOC medium at room 18. 19.E 2eeteSeeeg7eaagemeSaapean12-6hor REFERENCES of E.cof by high voltag 1p
Chapter:1 Protocol:26 Transformation of <i>E. coli</i> by Electroporation CHAPTER 1 > PROTOCOL 26 printer friendly version Protocol 26 Transformation of E. coli by Electroporation Electrocompetent bacteria are prepared by growing cultures to mid-log phase, washing the bacteria extensively at low temperature, and then resuspending them in a solution of low ionic strength containing glycerol. DNA is introduced during exposure of the bacteria to a short high-voltage electrical discharge. 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 Glycerol (10% v/v) (molecular biology grade), ice cold Pure H2O Milli-Q or equivalent, sterilized by filtration through prerinsed 0.45-µm filters. Store at 4°C. Nucleic Acids Plasmid DNA (recombinant plasmid) Construct using one of the methods described in Chapter 1, Protocol 17 , Chapter 1, Protocol 18 , Chapter 1, Protocol 19 , Chapter 1, Protocol 20 , Chapter 1, Protocol 21 and Chapter 1, Protocol 22 . Media GYT medium, ice cold LB medium, prewarmed to 37°C SOB agar plates containing 20 mM MgSO4 and the appropriate antibiotic SOC medium METHOD 1. Inoculate a single colony of E. coli from a fresh agar plate into a flask containing 50 ml of LB medium. Incubate the culture overnight at 37°C with vigorous aeration (250 rpm in a rotary shaker). 2. Inoculate two aliquots of 500 ml of prewarmed LB medium in separate 2-liter flasks with 25 ml of the overnight bacterial culture. Incubate the flasks at 37°C with agitation (300 cycles/minute in a rotary shaker). Measure the OD600 of the growing bacterial cultures every 20 minutes. 3. When the OD600 of the cultures reaches 0.4, rapidly transfer the flasks to an ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next step, place the centrifuge bottles in an ice-water bath. 4. Transfer the cultures to ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 15 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 500 ml of ice-cold pure H2O. 5. Harvest the cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 250 ml of ice-cold 10% glycerol. 6. Harvest the cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 20 minutes at 4°C. Decant the supernatant and resuspend the pellet in 10 ml of ice-cold 10% glycerol. 7. Harvest cells by centrifugation at 1000g (2500 rpm in a Sorvall GSA rotor) for 20 minutes at 4°C. Carefully decant the supernatant and use a Pasteur pipette attached to a vacuum line to remove any remaining drops of buffer. Resuspend the pellet in 1 ml of ice-cold GYT medium. 8. Measure the OD600 of a 1:100 dilution of the cell suspension. Dilute the cell suspension to a concentration of 2 x 1010 to 3 x 1010 cells/ml (1.0 OD600 = approx. 2.5 x 108 cells/ml) with ice-cold GYT medium. 9. Transfer 40 µl of the suspension to an ice-cold electroporation cuvette (0.2-cm gap) and test whether arcing occurs when an electrical discharge is applied (please see Step 16 below). If so, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low (<5 mEq). 10. To use the electrocompetent cells immediately, proceed directly to Step 12. Otherwise, store the cells at -70°C until required. For storage, dispense 40-µl aliquots of the cell suspension into sterile, ice-cold 0.5-ml microfuge tubes, drop into a bath of liquid nitrogen, and transfer to a -70°C freezer. 11. To use frozen electrocompetent cells, remove an appropriate number of aliquots of cells from the -70°C freezer. Store the tubes at room temperature until the bacterial suspensions are thawed and then transfer the tubes to an ice bath. 12. Pipette 40 µl of the freshly made (or thawed) electrocompetent cells into ice-cold sterile 0.5-ml microfuge tubes. Place the cells on ice, together with an appropriate number of bacterial electroporation cuvettes. 13. Add 10 pg to 25 ng of the DNA to be electroporated in a volume of 1-2 µl to each microfuge tube and incubate the tube on ice for 30-60 seconds. Include all of the appropriate positive and negative controls. 14. Set the electroporation apparatus to deliver an electrical pulse of 25 µF capacitance, 2.5 kV, and 200 ohm resistance. 15. Pipette the DNA/cell mixture into a cold electroporation cuvette. Tap the solution to ensure that the suspension of bacteria and DNA sits at the bottom of the cuvette. Dry condensation and moisture from the outside of the cuvette. Place the cuvette in the electroporation device. 16. Deliver a pulse of electricity to the cells at the settings indicated above. A time constant of 4-5 milliseconds with a field strength of 12.5 kV/cm should register on the machine. 17. As quickly as possible after the pulse, remove the electroporation cuvette and add 1 ml of SOC medium at room temperature. 18. Transfer the cells to a 17 x 100-mm or 17 x 150-mm polypropylene tube and incubate the cultures with gentle rotation for 1 hour at 37°C. 19. Plate different volumes (up to 200 µl per 90-mm plate) of the electroporated cells onto SOB agar medium containing 20 mM MgSO4 and the appropriate antibiotic. 20. Store the plates at room temperature until the liquid has been absorbed. 21. Invert the plates and incubate them at 37°C. Transformed colonies should appear in 12-16 hours. REFERENCES 1. Chassy B.M. and Flickinger J.L. 1987. Transformation of Lactobacillus casei by electroporation. FEMS Microbiol. Lett. 44:173-177. 2. Chassy B.M., Mercenier A., and Flickinger J. 1988. Transformation of bacteria by electroporation. Trends Biotechnol. 6:303-309. 3. Dower W.J., Miller J.F., and Ragsdale C.W. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 16:6127-6145. http://www.molecularcloning.com/members/protocol.jsp?pronumber=26&chpnumber=1 (1 / 2) [2002-2-18 16:15:27]
@片Molecular Cloning L A BO R A T OR CHAPTER1>PROTOCOL27 printer friendy versior Protocol 27 ing Bacterial ing X-gal a IPTG: anhgheppepiaeantiot octors and Ba .p METHOD into7-m a 45'C he they a ate8ompeea8068,8le8e5ernaatmo30o。 in the table below.Close h Components for Top Agar Size of Plate Molten Top Agar IPTG 0 y pour the molten top priate antbiotic and ry wid-type hese colonies are REFERENCES J.and Jacob F.1968.Genetic mapping of the regulator and operator genes of the c operon.J Mol.Biol printer friondly version 22e srtp://www.moleeularelaning.com/members/pratecol.==l [2902-2-18 16:15:34
Chapter:1 Protocol:27 Screening Bacterial Colonies Using X-gal and IPTG: ±-Complementation CHAPTER 1 > PROTOCOL 27 printer friendly version Protocol 27 Screening Bacterial Colonies Using X-gal and IPTG: -Complementation -complementation occurs when two inactive fragments of E. coli -galactosidase associate to form a functional enzyme. Many plasmid vectors carry a short segment of DNA containing the coding information for the first 146 amino acids of -galactosidase. Vectors of this type are used in host cells that express the carboxy-terminal portion of the enzyme. Although neither the host nor the plasmid-encoded fragments of -galactosidase are themselves active, they can associate to form an enzymatically active protein. Lac+ bacteria that result from -complementation are easily recognized because they form blue colonies in the presence of the chromogenic substrate X-gal. However, insertion of a fragment of foreign DNA into the polycloning site of the plasmid almost invariably results in production of an aminoterminal fragment that is no longer capable of -complementation. Bacteria carrying recombinant plasmids therefore form white colonies. The development of this simple blue-white color test has greatly simplified the identification of recombinants constructed in plasmid vectors. 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 IPTG solution (20% w/v) X-gal solution (2% w/v) Media Rich broth agar plates containing the appropriate antibiotic Rich broth top agar Vectors and Bacterial Strains E. coli culture, transformed with recombinant plasmids Use bacteria transformed by one of the methods described in Chapter 1, Protocol 23 , Chapter 1, Protocol 24 , Chapter 1, Protocol 25 , Chapter 1, Protocol 26 . METHOD 1. Dispense aliquots of molten top agar into 17 x 100-mm tubes. Place the tubes in a 45°C heating block until they are needed. 2. Remove the first tube from the heating block. Working quickly, add 0.1 ml of bacterial suspension containing <3000 viable bacteria for a 90-mm plate and <10,000 for a 150-mm plate. Close the top of the tube and invert it several times to disperse the bacteria through the molten agar. 3. Open the tube and add the appropriate amounts of X-gal and IPTG (if required) as shown in the table below. Close the top of the tube and gently invert it several times to mix the contents. Components for Top Agar Amount of Reagent Size of Plate Molten Top Agar X-gal IPTGa 90 mm 3 ml 40 µl 7 µl 150 mm 7 ml 100 µl 20 µl aMay not be required; please see the entry on IPTG in the Materials list. 4. Quickly pour the molten top agar into the center of a hardened agar plate containing the appropriate antibiotic and distribute the solution by swirling. 5. Repeat Steps 2-4 until all of the samples have been plated. 6. Allow the soft agar to harden at room temperature, wipe any condensation from the lid of the plates, and then incubate the plates in an inverted position for 12-16 hours at 37°C. 7. Remove the plates from the incubator and store them for several hours at 4°C, to allow the blue color to develop. 8. Identify colonies carrying recombinant plasmids. • Colonies that carry wild-type plasmids contain active -galactosidase. These colonies are pale blue in the center and dense blue at their periphery. • Colonies that carry recombinant plasmids do not contain active -galactosidase. These colonies are creamy-white or eggshell blue, sometimes with a faint blue spot in the center. 9. Select and culture colonies carrying recombinant plasmids. REFERENCES 1. Davies J. and Jacob F. 1968. Genetic mapping of the regulator and operator genes of the lac operon. J. Mol. Biol. 36:413-417. 2. Ullmann A., Jacob F., and Monod J. 1967. Characterization by in vitro complementation of a peptide corresponding to an operator-proximal segment of the beta-galactosidase structural gene of Escherichia coli. J. Mol. Biol. 24:339-343. 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=27&chpnumber=1 [2002-2-18 16:15:34]