ISpring Harber laboratoy hesMokar Cmn.Chapter Target DNA is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent E coi. The maximum number of correct clones can generally be obtained from igation reactions containing equimolar amounts of plasmid and target DNAs, with the total DNA concentration being <100 pg/ml Blunt-end igation catalyzed by bacteriophage T4 DNA ligase is suppressed by high concentrations (5 mM) of ATP and Protocol 20. Dephosphorylation of Plasmid DNA ring ligation in vitro, T4 DNA lgase will catalyze the fomation of a phosphodiester bond between adjacent nucleotides only if one nucleotide camies a 5-phosphate residue and the other caries a 3-hydroxyl terminus. Recircularization of vector DNA can therefore be minimized by removing the 5 -phosphate residues from both termini af the linear, double- tranded plasmid DNA with alkaline phosphatase Protocol 21: Addition of Synthetic Linkers to Blunt-ended DNA Linkers are smal seif-complementary pieces of synthetic DNA, usualy 8-16 nucleotides in length, that anneal to form blunt-ended, double-stranded molecules containing a restriction site Inkers are used to equip blunt-ended temin of DNA with restricion sites as an aid to doning Protocol 22 Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose igation in ow -meling temperature agarose is much less eficient than igation with punted DNA in fee solufion and requires a large amount of DNA ligase. The method is used chiefly for rapid subdoning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs Protocol 23: The Hanahan Wethod for Preparation and Transformation of Competent E col: High-efficiency Transformation This procedure generates competent cultures of E coi that can be transformed at high frequencies(5x 105 transformed colonies/ mg of superhelical plasmid DNA). IMPORTANT AI steps in this protocol should be camed ouf aseptically Protocol 24: The Inoue Method for Preparation and Transformation of Competent E Col: " Ultra-Competent" Cells This protocol reproducibly generates competent ctures of E cof that yield 1x 10t 3 x 108 transformed colonies mg of plasmid DNA The protocal works optimaly when the bacterial ture is grown at 18 C. ifa suitable incubator is not available, a standard bacterial shaker can be set up na 4 Coold room and regulated to 18C 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 5x 10 to 2 x 10 transformed colonies/g of supercoiled plasmid DNA. Protocol 26: Transformation of E coli by Electroporation Eedtrocompetent bactenia are prepared by growng atures to mid-og phase, washing the bacteia 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- Protocol 27: Screening Bacterial Colonies Using X-gal and IPTG: a-Complementation to form a functional enzyme. Many plasmid vectors carry a shor segment of DNA containing the coding information for the first 146 amino acids of F-galactosidase Vedors of his type are used host cells that express the carboxy-terminal portion of the enzyme. Although neither the host nar the plasmid-encoded fragments of galactosidase are themselves active, they can associate to form an enzymatically active protein. Lac bacteria that result fomo complementation are easly recogn zed because they fom 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 amino-terminal ment that is no longer capable of a-complementation. Bacteria carrying recombinant plasmids therefore form white colonies. The development of this simple blue-nthite color test has greatly simplified the identification of recombinants onshuded in plasmid vectors. Protocol 28: Screening Bacterial Colonies by Hybridization: Small Numbers This procedure, a variant of the Grunstein and Hogness( 1979) method, is used to screen a smal number of bacterial colonies(< 200) that are dispersed over several agar plates and are to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar plate. After a penod f growth, the colonies on the filter are lysed and processed for hybridization. The master plate is stored unti the results of the screening procedure become available Protocol 29. Screening Bacterial Colonies by Hybridization: Intermediate Numbers Bacterial olonies growing an agar plates are transferred en masse to nitrocellulose filters. The patial arrangement of colonies on the pla es is preserved an the fters. After transfer, the fiters are processed for hybridization to an appropriate radiolabeled probe while the origina master plate is incubated for a few hours to allow the bacterial colonies to regrow in their riginal posifions. This technique, a variant of the Grunstein and Hogness(1975) method, was developed at Cold Spring Harbor Laboratory in 1975. The procedure warks best with 90-mm plates containing <2500 colonies Protocol 30. Screening Bacterial Colonies by Hybridization: Large Numbers This procedure is used to plate, replicate, and subsequenty screen large numbers of bacterial colonies ( up to 2x 10 oolonies per 150-mm plate or 10 colonies per 90-mm plate) Protocol 31: Lysing Colonies and Binding of DNA to Filters In this protocol, based on the procedure of Grunstein and Hogness(1975), alkal is used to berate DNA ram bacterial colonies an nitrocellulose or nylon fiters. The DNA is then fxed to the filter by UV-eross-inking ar baking under vacum. Protocol 32 Hybridization of Bacterial DNA on Filters This protocol describes procedures to hybidize DNA from transformed colonies immobilized on iters with radiolabeled probes and to reoover fram a master plate the corresponding colonies that hybridize specificaly to the probe. The method is based an the procedure published by Grunstein and Hogness(1975) 坤 wwnoeadarcbnig com/merberschapter sp?cha=11232012t母Cold Spring Harbor Laboratory Press - Molecular Cloning - Chapter 1 Target DNA is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent E. coli. The maximum number of "correct" clones can generally be obtained from ligation reactions containing equimolar amounts of plasmid and target DNAs, with the total DNA concentration being <100 µg/ml. Blunt-end ligation catalyzed by bacteriophage T4 DNA ligase is suppressed by high concentrations (5 mM) of ATP and polyamines such as spermidine. Protocol 20: Dephosphorylation of Plasmid DNA During ligation in vitro, T4 DNA ligase will catalyze the formation of a phosphodiester bond between adjacent nucleotides only if one nucleotide carries a 5´-phosphate residue and the other carries a 3´-hydroxyl terminus. Recircularization of vector DNA can therefore be minimized by removing the 5´-phosphate residues from both termini of the linear, double￾stranded plasmid DNA with alkaline phosphatase. Protocol 21: Addition of Synthetic Linkers to Blunt-ended DNA Linkers are small self-complementary pieces of synthetic DNA, usually 8-16 nucleotides in length, that anneal to form blunt-ended, double-stranded molecules containing a restriction site. Linkers are used to equip blunt-ended termini of DNA with restriction sites as an aid to cloning. Protocol 22: Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs. Protocol 23: The Hanahan Method for Preparation and Transformation of Competent E. coli: High-efficiency Transformation This procedure generates competent cultures of E. coli that can be transformed at high frequencies (5 x 108 transformed colonies/mg of superhelical plasmid DNA). IMPORTANT All steps in this protocol should be carried out aseptically. 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. 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. 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. 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 amino-terminal 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. Protocol 28: Screening Bacterial Colonies by Hybridization: Small Numbers This procedure, a variant of the Grunstein and Hogness (1979) method, is used to screen a small number of bacterial colonies (<200) that are dispersed over several agar plates and are to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar plate. After a period of growth, the colonies on the filter are lysed and processed for hybridization. The master plate is stored until the results of the screening procedure become available. Protocol 29: Screening Bacterial Colonies by Hybridization: Intermediate Numbers Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the filters are processed for hybridization to an appropriate radiolabeled probe while the original (master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their original positions. This technique, a variant of the Grunstein and Hogness (1975) method, was developed at Cold Spring Harbor Laboratory in 1975. The procedure works best with 90-mm plates containing <2500 colonies. Protocol 30: Screening Bacterial Colonies by Hybridization: Large Numbers This procedure is used to plate, replicate, and subsequently screen large numbers of bacterial colonies (up to 2 x 104 colonies per 150-mm plate or 104 colonies per 90-mm plate). Protocol 31: Lysing Colonies and Binding of DNA to Filters In this protocol, based on the procedure of Grunstein and Hogness (1975), alkali is used to liberate DNA from bacterial colonies on nitrocellulose or nylon filters. The DNA is then fixed to the filter by UV-cross-linking or baking under vacuum. Protocol 32: Hybridization of Bacterial DNA on Filters This protocol describes procedures to hybridize DNA from transformed colonies immobilized on filters with radiolabeled probes and to recover from a master plate the corresponding colonies that hybridize specifically to the probe. The method is based on the procedure published by Grunstein and Hogness (1975). http://www.molecularcloning.com/members/chapter.jsp?chapter=112 (2 / 3) [2002-2-18 16:10:49]