How does BP Clonase work?
Gateway BP Clonase Enzyme Mixes Gateway BP Clonase enzyme contains both Int (Integrase) and IHF (Integration Host Factor) proteins that catalyze the in vitro recombination of PCR products or DNA segments from clones (containing attB sites) and a Donor vector (containing attP sites) to generate Entry clones.
What does LR Clonase do?
Gateway™ LR Clonase™ II enzyme mix catalyzes in vitro recombination between an entry clone (atL-flanked “gene”) and an atR-containing destination vector to generate an atB- containing expression clone. coli and “gene” represents any DNA segment of interest (e.g. PCR product, cDNA, genomic DNA).
What is a BP reaction?
The BP Reaction is a recombination reaction which is explained in the following lines. For the reaction to take place, the gene of interest is amplified with the help of an attB tagged primer pair. The donor vector includes attP sites. The resulting entry clone contains the gene of interest flanked by attL sites.
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 att recombination site?
Both HK and ΦC31 systems comprise attB/attP attachment sites that serve as points of recombination, and the recombinases that catalyze recombination. In each family, DNA exchange requires host-encoded proteins for recombination that differ between systems.
How do you do LR reactions?
LR reaction
- Add the following components to a 1.5 ml tube at room temperature and mix:
- Thaw on ice the Invitrogen LR Clonase II enzyme mix for about 2 minutes.
- 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 a TOPO vector?
TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. For “blunt end” TOPO cloning, the recipient vector does not have overhangs and blunt-ended DNA fragments can be cloned.
What is ATT site lambda phage?
The minimal phage att site is composed of approximately 240-base pairs and four distinct binding sites for Int protein, at least three of which are crucial for function. This ‘donor site’ recombines efficiently with a smaller ‘recipient site’ that lacks the extensive interactions with Int protein.
What is phage attachment site?
This is where on the surface of a bacterium that a phage recognizes in the course of adsorption. In terms of virion attachment to a bacterial surface, this event can be either reversible or irreversible, depending on the phage involved, with the reversible step, of course, preceding the irreversible step.
How do you mix BP Clonase 2?
Vortex the BP Clonase II enzyme mix briefly twice (2 seconds each time). To each sample (Step 1, above), add 2 µl of BP Clonase II enzyme mix to the reaction and mix well by vortexing briefly twice. Microcentrifuge briefly.
What is in Gateway BP Clonase II?
Gateway® BP 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™ BP Clonase™ II enzyme mix includes proteinase K solution (2 µg/µl) and a positive control vector. Store at -20°C or -80°C.
What is the recommended storage temperature for Gateway BP Clonase II?
Gateway™ BP Clonase™ II enzyme mix includes proteinase K solution (2 µg/µl) and a positive control vector. Store at -20°C or -80°C. Guaranteed stable for 6 months when properly stored.
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. Microcentrifuge briefly.