What does LR Clonase do?
Mediating the recombination reaction This reaction is mediated by a robust enzyme mixture called LR Clonase™, which contains the necessary protein activity to excise the gene of interest from the Entry clone and integrate it into the Destination vector, which then becomes your expression clone (Figure 1).
How does BP Clonase work?
The BP clonase enzyme mix recombines attB sites with attP sites, generating attL and attR sites; whereas the LR clonase enzyme mix catalyzes the reverse reaction (Fig. 1A). Each recombination event is precise, with no nucleotides either gained or lost.
What is BP reaction in Gateway cloning?
The BP Reaction takes place between the attB sites flanking the insert and the attP sites of the donor vector. This reaction is catalyzed by the BP Clonase enzyme mix and generates the entry clone containing the DNA of interest flanked by attL sites.
What is an entry clone?
An Entry clone contains your gene of interest flanked by attL sequences, which are then used to recombine with attR sequences to create your desired expression clone.
What is LR reaction?
The LR Reaction, again is a recombination reaction between attL and attR sites. The reaction generates an expression clone and is catalyzed by recombinant proteins. Recombination between these sites generates two molecules. One molecule contains the DNA segment of interest, the other molecule is a by-product.
What is Gateway in biology?
The Gateway cloning System, invented and commercialized by Invitrogen since the late 1990s, is a molecular biology method that enables researchers to efficiently transfer DNA-fragments between plasmids using a proprietary set of recombination sequences, the “Gateway att” sites, and two proprietary enzyme mixes, called …
What is a gateway plasmid?
What is an attB site?
The attB site is a short DNA sequence (less than 30 bp) corresponding to the crossover region at which strand exchange takes place (7). Two imperfect inverted repeats that bind to the integrase surround a 7-bp overlap region delimited by the scattered cuts made by the recombinase.
What is a Golden Gate reaction?
The Golden Gate cloning is an in vitro molecular cloning method that is based on the enzymatic capability of Type IIs restriction enzymes. This methodology enables simultaneous and directional assembly of multiple DNA fragments in a single-tube reaction.
How do I use the Invitrogen LR Clonase II enzyme?
Thaw on ice the Invitrogen LR Clonase II enzyme mix for about 2 minutes. Vortex the LR Clonase II enzyme mix briefly twice (2 seconds each time). To each sample (Step 1, above), add 2 µl of LR Clonase II enzyme mix to the reaction and mix well by vortexing briefly twice.
What is the storage temperature for LR Clonase II?
Gateway® LR Clonase® II contains enzymes and buffer in a single mix to enable convenient ten-microliter reaction set-up with fewer pipetting steps. For Research Use Only. Not for use in diagnostic procedures. Gateway™ LR Clonase™ II enzyme mix includes proteinase K solution (2 µg/µl) and a positive control vector. Store at -20°C or -80°C.
What are the ingredients in the gateway® LR Clonase® enzyme kit?
All Gateway® LR Clonase® enzyme kits include proteinase K solution (2 µg/µl) and a positive control vector. Store LR Clonase® enzyme at -80°C; LR Clonase® II or II Plus Enzyme mixtures at -20°C. Guaranteed stable for 6 months when properly stored.
How does Invitrogen™ Gateway™ cloning work?
As explained, the core of Invitrogen™ Gateway™ cloning is the Entry vector. Once the Entry clone is ready, the gene of interest is easily shuttled to a secondary plasmid, the Destination vector. This reaction is mediated by a robust enzyme mixture called LR Clonase™, which contains the necessary protein activity…