What is a BP reaction?
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 are att sequences?
The DNA segments flanking the recombination sites are switched, such that after recombination, the att sites are hybrid sequences comprised of sequences donated by each parental vector. For example, attL sites are comprised of sequences from attB and attP sites.
What is BP cloning?
Gateway® Cloning Mechanism (a)The BP Reaction (PCR fragment + Donor vector = Entry Clone) 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.
How is Benchling used in cloning?
Hit cmd/ctrl + C to copy this region. Open your second sequence file and find the enzyme cut sites and shift-select the region between them. Paste the copied region by hitting cmd/ctrl + V. Note: Benchling will automatically reverse the fragment, if needed, and handle ligating sticky ends.
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 the 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 an Attl site?
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.
How do you do Gibson on Benchling?
To access the Assembly Wizard, first open a sequence file. At the bottom of your screen you will find the Assembly Wizard next to Split Workspace. Click Assembly Wizard, then select Create New Assembly. In the options provided, select Gibson and press Start to proceed with the assembly.
Is topoisomerase used in PCR?
When the free 5′ ends of the PCR product strands attach to the topoisomerase 3′ end of each vector strand, the strands are covalently linked by the already bound topoisomerase. This reaction proceeds efficiently when this solution is incubated at room temperature with required salt.
What is the BP reaction in DNA extraction?
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. As a byproduct of the reaction, the ccdB gene is excised from the donor vector.
How do I perform a BP Clonase reaction?
To perform the BP clonase reaction, select the pDONR221 and DTU76545 PCR Product sequences then go Cloning → Gateway Cloning. The Gateway cloning tool will identify the att sites present on both the vector and the PCR product insert, confirm they are appropriately oriented, and inform that a BP reaction will be performed.
How do I perform the BP and LR reactions using the gateway?
To simplify your Gateway reactions it is possible to use the Gateway cloning tool to perform the BP and LR reactions in a single operation. Simply select your PCR product insert with attB sites, your pDONR vector and your pDest vector and go Cloning → Gateway Cloning.
What is the difference between LR and BP reactions?
The LR Reaction takes place between the attL sites of the generated entry clone and the attR sites of the destination vector. This reaction is catalyzed by the LR Clonase enzyme mix. As a result, an expression clone with the DNA of interest flanked by attB sites is generated. As in the BP reaction, a DNA fragment containing