T4 DNA ligase

Product Contents Quality Control Assays Promega Corporation 2800 Woods Hollow Road Madison, WI 53711-5399 USA Telephone 608-274-4330 Toll Free 800-356-9526 Fax 608-277-2516 Internet www.promega.com PRODUCT USE LIMITATIONS, WARRANTY, DISCLAIMER Promega manufactures products for a number of intended uses. Please refer to the product label for the intended use statements for specific products. Promega products contain chemicals which may be harmful if misused. Due care should be exercised with all Promega products to prevent direct human contact. Each Promega product is shipped with documentation stating specifications and other technical information. Promega products are warranted to meet or exceed the stated specifications. Promega's sole obligation and the customer's sole remedy is limited to replacement of products free of charge in the event products fail to per- form as warranted. 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Please visit our Web site for more information. Packagene, pGEM and Wizard are registered trade- marks of Promega Corporation. Coomassie is a registered trademark of Imperial Chemical Industries. All specifications are subject to change without prior notice. Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products. T4 DNA Ligase Blue/White Cloning Qualified T4 DNA Ligase: Size Part No. (Weiss units) M180A 100 M180B 500 M179A (High Conc.) 500 Ligase Buffer, 10X (C126A, C126B): The Ligase 10X Buffer supplied with this enzyme has a composition of 300mM Tris-HCl (pH 7.8), 100mM MgCl2, 100mM DTT and 10mM ATP. The performance of this buffer depends on the integrity of the ATP. SSttoorree tthhee bbuuffffeerr iinn ssmmaallll aalliiqquuoottss aatt ––2200°°CC ttoo mmiinniimmiizzee ddeeggrraaddaattiioonn ooff tthhee AATTPP aanndd DDTTTT. Note: The DTT in the Ligase 10X Buffer may precipitate upon freezing. If this occurs, vortex the buffer until the precipitate is in solution (typically 1–2 minutes). The performance of the product is not affected provided that the precipitate is resuspended. Enzyme Storage Buffer: T4 DNA Ligase is supplied in 10mM Tris-HCl (pH 7.4), 50mM KCl, 1mM DTT, 0.1mM EDTA and 50% glycerol. Source: E. coli strain expressing a recombinant clone. Unit Definition: 0.01 Weiss unit of T4 DNA Ligase is defined as the amount of enzyme required to catalyze the ligation of greater than 95% of the Hind III fragments of 1µg of Lambda DNA at 16°C in 20 minutes. See the unit concentration on the Product Information Label. Storage Temperature: Store at –20°C. Avoid multiple freeze-thaw cycles and exposure to frequent temperature changes. See the expiration date on the Product Information Label. Activity Assays Blue/White Assay: pGEM®-3Zf(+) Vector is digested with representative restriction enzymes (leaving 5´-termini, 3´-termini or blunt ends). Each microgram of cut plasmid is ligated with 4 units of T4 DNA Ligase. The DNA is then transformed into JM109 cells that are plated on X-Gal/IPTG/Ampicillin plates. White colonies result from transformation with ligated plasmids with damaged ends. These white colonies represent the number of false positives expected in a typical cloning experiment. Enzymes that generate overhangs must produce fewer than 2% white colonies, and blunt-cutting enzymes must produce fewer than 5% white colonies. Contaminant Activity Endonuclease Assay: To test for endonuclease activity, 1µg of Type I supercoiled plasmid DNA is incubated with 20 units of T4 DNA Ligase in 1X Ligase Buffer (Stock# C126 at 1X) for 16 hours at 37°C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel. There must be no visible nicking or cutting of the DNA. Single-Stranded and Double-Stranded DNase Assay: To test for DNase activity, 50ng of radiolabeled single-stranded or double-stranded DNA is incubated with 20 units of T4 DNA Ligase in 1X Ligase Buffer (Stock# C126 at 1X) for 16 hours at 37°C. Minimum passing specification is <2% release of single-stranded and <1% release of double-stranded radiolabeled nucleotides as monitored by scintillation counting of TCA-soluble material. RNase Assay: To test for RNase activity, 50ng of radiolabeled RNA is incubated with 20 units of T4 DNA Ligase in 1X Ligase Buffer (Stock# C126 at 1X) for 5 hours at 37°C. Minimum passing specification is <3% release of radiolabeled nucleotides as monitored by scintillation counting of TCA-soluble material. Physical Purity: The purity is ≥90% as judged by SDS-polyacrylamide gels with Coomassie® blue staining. Part# 9PIM180 Revised 1/07 Part# 9PIM180 Printed in USA. Revised 1/07 A F 9 P I M1 8 0 0 1 0 7 M1 8 0 Promega Corporation · 2800 Woods Hollow Road·Madison, WI 53711-5399 U.S.A. · Toll Free in the USA 800-356-9526 · Telephone 608-274-4330 · Internet www.promega.com Usage Information I. Description T4 DNA Ligase catalyzes the joining of two strands of DNA between the 5´-phosphate and the 3´-hydroxyl groups of adjacent nucleotides in either a cohesive-ended or blunt-ended configuration (1). The enzyme has also been shown to catalyze the joining of RNA to either a DNA or RNA strand in a duplex molecule but will not join single- stranded nucleic acids (1). II. Standard Applications A. Ligation of DNA Material to Be Supplied by the User • Nuclease-Free Water (Cat.# P1193) We recommend using a 1:1, 1:3 or 3:1 molar ratio of vector:insert DNA when cloning a fragment into a plasmid vector. These ratios will vary with other types of vectors, for example, cDNA and genomic cloning vectors. The following example illustrates the conversion of molar ratios to mass ratios for a 3.0kb plasmid and a 0.5kb insert DNA fragment. ng of vector × kb size of insert × molar ratio of insert = ng of insert kb size of vector vector Example: How much 0.5kb insert DNA should be added to a ligation in which 100ng of 3kb vector will be used? The desired vector:insert ratio will be 1:3. 100ng vector × 0.5kb insert × 3 = 50ng insert 3kb vector 1 The following ligation reaction of a 3kb vector and a 0.5kb insert DNA uses a 1:1 vector:insert ratio. Typical ligation reactions use 100–200ng of vector DNA. 1. Assemble the following reaction in a sterile microcentrifuge tube: vector DNA 100ng insert DNA 17ng Ligase 10X Buffer 1µl T4 DNA Ligase (Weiss units) 0.1–1u Nuclease-Free Water to final volume of 10µl 2. Incubate the reaction at: room temperature for 3 hours, or 4°C overnight, or 15°C for 4–18 hours. Notes: 1. There is considerable latitude in the temperature and time needed for successful ligations. The optimal temperature for a ligation is a balance between the optimal temperature for T4 DNA Ligase enzyme activity (25°C) (1) and the temperature necessary to ensure annealing of the fragment ends, which can vary with the length and base composition of the overhangs. Shorter duplexes (linkers less than 16 bases long) require lower temperatures as a result of their lower melting temperatures (Tm). In general, ligation reactions performed at lower temperatures require longer incubation times. The scientific literature reflects this variability in ligation conditions. Blunt-end ligations generally are efficient at temperatures between 15–20°C for 4–18 hours, while sticky ends are ligated effectively at room temperature (22°C) for 3 hours or 4–8°C overnight. 2. The ligation conditions given in this protocol are based on the conditions used at Promega for quality control of lambda vectors with sticky ends. These ligation condi- tions have been developed using Promega Blue/White Cloning-Qualified T4 DNA Ligase. 3. The addition of polyethylene glycol (PEG) to ligation reactions can promote ligation of blunt-ended fragments by “macromolecular crowding” (2). We do not recommend the use of PEG in ligations, however, due to extreme variability in the quality of PEG. In addition, the use of PEG can lead to undesirable concatemerization when cloning cDNAs, and residual PEG is inhibitory to lambda packaging reactions. III. Additional Information Molecular Weight: 68kDa (3). Requirements: Mg2+, ATP and DTT (3). The optimum concentration of Mg2+ is 10mM. Mn2+ may be substituted for Mg2+ but is only 25% as effective as Mg2+ (1). Inhibition: 50% inhibition by greater than 150mM NaCl (activity measured at nicks) (3). Other inhibitors include 0.2M K+, Cs+, Li+, NH4+ and 1mM spermine (1). Inactivation: Heat to 70°C for 10 minutes (4). IV. References 1. Engler, M.J. and Richardson, C.C. (1982) In: The Enzymes, Boyer, P.D., ed., Academic Press, New York, NY. 2. Zimmerman, S.B. and Pheiffer, B.H. (1983) Macromolecular crowding allows blunt-end ligation by DNA ligases from rat liver or Escherichia coli. Proc. Natl. Acad. Sci. USA 8800, 5852–6. 3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 4. Protocols and Applications Guide, Third Edition (1996) Promega Corporation. Part# 9PIM180 Printed in USA. Revised 1/07