TtAgo is a prokaryotic argonaute from the Gram-negative, thermophilic bacterium Thermus thermophilus which functions as a DNA-guided endonuclease when provided with a 16–18 nucleotide long 5′-phosphorylated single-stranded DNA oligonucleotide guide. The RNase H-like active site of TtAgo requires divalent Mg2+ (or Mn2+) (See product usage note #1)metal ions for the reaction to occur and cleaves a complementary substrate between the bases corresponding to positions 10 and 11 of the DNA guide.TtAgo mechanism of actionTtAgo is active from 65–85°C (See product usage note #2) and exhibits high activity on single-stranded DNA, moderate to high activity on double-stranded DNA, and mild activity on single-stranded RNA substrates. TtAgo does not introduce double-strand breaks in double-stranded DNA substrates when provided with a single guide, and instead only introduces a nick in the strand complementary to the guide. Activity is not observed on double-stranded RNA substrates. The corresponding 5? and 3? ends of cleavage products are compatible with downstream ligation.(1) It has been demonstrated that in some cases, the addition of thermostable single-stranded DNA binding protein (ET SSB, NEB #M2401) and/or a thermostable helicase can aid in the cleavage of some dsDNA substrates.In the absence of a guide, the apo-form of TtAgo can degrade dsDNA through a process known as “chopping,” which is thought to be the mechanism by which TtAgo naturally generates guides from invading or foreign DNA.(2)To prevent this non-specific activity, guide should be kept in an approximately 5X molar excess over TtAgo. Additionally, a pre-loading step can be performed in which guide is incubated with TtAgo at ~75°C for 10-30 mins to form the nucleoprotein complex prior to substrate addition. Please see the Protocols, Manuals & Usage section for more details.TtAgo is recommended for in vitro applications.For larger volume or higher concentration requirements, customized and bulk packaging is available by purchasing through the Customized Solutions department at NEB. Please contact custom@neb.com for further information.
Product Source
Thermus thermophilus argonaute (TtAgo) is purified from an E. coli strain that carries a cloned gene from the Gram-negative, thermophilic bacterium Thermus thermophilus which is expressed as a recombinant N-terminal 6X His-tagged fusion.
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