Cell Signaling Product TSAP Thermosensitive Alkaline Phosphatase Size Cat.# Price (Fr) TSAP Thermosensitive Alkaline Phosphatase For Laboratory Use. 100 units M9910 116.00 Description: TSAP Thermosensitive Alkaline Phosphatase catalyzes the removal of 5´ phosphate groups from DNA, thus preventing the recircularization and religation of linearized cloning vector DNA during ligation. It is effective on 3´ overhangs, 5´ overhangs and blunt ends. It is also useful for preparing DNA for 5´ end-labeling by removing existing phosphate groups from the 5´ end. TSAP is irreversibly inactivated by heating at 74°C for 15 minutes. Therefore, a DNA cleanup step is not required before proceeding to a ligation reaction. TSAP is fully active in all restriction enzyme reaction buffers tested under the conditions listed below, facilitating a streamlined restriction digestion, dephosphorylation and ligation reaction. Features: • Easy To Use: TSAP is active in all Promega restriction enzyme buffers, eliminating any cleanup steps or buffer swaps. • Convenient: TSAP is irreversibly inactivated by heating at 74°C for 15 minutes. This allows streamlining of the restriction enzyme digestion, dephosphorylation and ligation procedure by eliminating the need for cleanup after alkaline phosphatase treatment. • Blue/White Cloning-Qualified: Promega’s blue/white cloning assay provides a higher level of quality control for enzymes used in cloning applications. • Provided with Promega MULTI-CORE™ Buffer. Storage Conditions: Store at –20°C. See the expiration date on the label. Protocol TSAP Thermosensitive Alkaline Phosphatase Protocol Comparison of Alkaline Phosphatases. Heat Inactivated Inactivation Temperature Incubation Time Special Buffer Required/Recommended Active in all Promega Restriction Enzyme Buffers Units required in different RE Buffers Blue/White Cloning-Qualified CIAP = Calf Intestinal Alkaline Phosphatase TSAP Yes 74 15 min No Yes 1–2 Yes CIAP* No N/A 2 ⋅ 30 min Yes No N/A Yes Only TSAP does not require a special buffer and is active in all Promega restriction enzyme buffers, making it the most convenient and cost-effective choice. 9493LA Part# 9PIM991 Polymerases DNA Polymerase I Product DNA Polymerase I For Laboratory Use. Description: DNA Polymerase I catalyzes the template-directed polymerization of nucleotides into duplex DNA in a 5´→3´ direction. DNA Polymerase I possesses a 3´→5´ exonuclease activity or “proofreading” function, which lowers the error rate during DNA replication, and also contains a 5´→3´ exonuclease activity, which enables the enzyme to replace nucleotides in the growing strand of DNA by nick translation. The enzyme, purified from recombinant E. coli, is capable of catalyzing de novo synthesis of synthetic homopolymers and provides a convenient method for the preparation of a variety of defined DNA substrates. Features: • Flexible: DNA Polymerase I may be used in a variety of molecular applications. • May Be Heat-Inactivated: DNA Polymerase I is inactivated by heating at 68°C for 10 minutes. • Provided with 10X Reaction Buffer: 500mM Tris-HCl (pH 7.2 at 25°C), 100mM MgSO4, 1mM DTT. Storage Conditions: Store at –20°C. Protocol DNA Polymerase I Protocol Part# 9PIM205 Product DNA Polymerase I Large (Klenow) Fragment Size Conc. Cat.# Price (Fr) DNA Polymerase I Large (Klenow) Fragment For Laboratory Use. Description: DNA Polymerase I Large (Klenow) Fragment is a DNA-dependent DNA polymerase that lacks the 5´→3´ exonuclease activity of intact E. coli DNA Polymerase I but retains its 5´→3´ polymerase, 3´→5´ exonuclease and strand displacement activities. The enzyme is a 68kDa C-terminal fragment of DNA Polymerase I. The 5´→3´ polymerase activity of Klenow Fragment can be used to fill in 5´-protruding ends with unlabeled or labeled dNTPs, to sequence single- or double-stranded DNA templates, for in vitro mutagenesis using synthetic oligonucleotides, for cDNA second-strand synthesis and to generate single-stranded DNA probes. The 3´→5´ exonuclease activity can be used to generate blunt ends from a 3´-overhang. Features: • Flexible: DNA Polymerase I Large (Klenow) Fragment may be used in a variety of molecular applications. It is also active in many Promega 1X restriction enzyme buffers. • May Be Heat-Inactivated: DNA Polymerase I Large (Klenow) Fragment is inactivated by heating at 75°C for 10 minutes. • Provided with 10X Reaction Buffer: 500mM Tris-HCl (pH 7.2 at 25°C), 100mM MgSO4, 1mM DTT. • Choose Your Configuration: Learn more about our custom options for this product at: www.promega.com/myway/ Storage Conditions: Store at –20°C. Protocol DNA Polymerase I Large (Klenow) Fragment Protocol Part# 9PIM220 150 u 5 u/µl M2201 155.00 500 u 5 u/µl M2206 440.00 Size Conc. Cat.# Price (Fr) 500 u 5–10 u/µl M2051 134.00 2,500 u 5–10 u/µl M2055 536.00 4 For complete and up-to-date product information visit: www.promega.com/catalog 89 Cloning and DNA Markers Pagina 92
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