What is pDONR221?
Detailed Vector Information: pDONR221 Description: Recombinational donor/master vector with M13 F and R primer sites; kanamycin resistance; recombinational cloning. Comments: The position of features were determined for the unrecombined (empty) form of the vector, which is described in detail on the Invitrogen website.
How do I create a gateway cloning primer?
Thermo Fisher Scientific recommend that to enable efficient Gateway® cloning, primers for addition of attB sites MUST contain the following structure: Four guanine (G) residues at the 5′ end of each primer, followed by a 25 bp attB site, and followed by At least 18-25 bp of template/gene-specific sequence.
What is the advantage of Gateway cloning?
Applications and Advantages of Gateway® Cloning High cloning efficiency. Reduced false positive rate due to the presence of ccdB gene. Flexibility in moving the gene of interest into and out of many vectors. Allowing to maintain desired reading frame and orientation.
Is Gateway cloning expensive?
Gateway cloning does take more time for initial set-up, and is more expensive than traditional restriction enzyme and ligase-based cloning methods, but it saves time, and offers simpler and highly efficient cloning for down-stream applications.
What is attB1 and attB2?
ATG. Table 1 provides examples of primer pairs (attB1 and attB2) which can be used to amplify genes for specific protein expression formats. Shine-Dalgarno (E. coli ribosome binding sequence) and Kozak (eukaryotic ribosome recognition sequence) show are typical sequences.
Who invented Gateway cloning?
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 attP and attB?
The attPλ is composed of two integrase binding sites (P and P′) and the core binding site (COC′). The attBλ site comprises a central overlap region, known also as O (7 bp), which is flanked by imperfect, inverted repeats, B (7 bp), and B′ (7 bp) (Mizuuchi and Mizuuchi, 1985).
What is the ccdB gene?
The ccdB gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin system encoded by the ccd operon, which is responsible for plasmid maintenance during cell division.
What is CcdB gene?
What does LR Clonase do?
Gateway™ LR Clonase™ II enzyme mix catalyzes the in vitro recombination between an entry clone (containing a gene of interest flanked by attL sites) and a destination vector (containing attR sites) to generate an expression clone.