![]() The first step starts with the BP reaction. Unlike standard Gateway cloning, multisite Gateway cloning utilizes the site-specificity of four different att sites to recombine multiple fragments into a single construct in one step. Multisite Gateway cloning can assemble multiple fragments into a single destination vector. In addition, numerous collections exist which contain thousands of sequenced cDNAs located between attL1 and attL2. Multiple destination vectors exist, which are engineered to achieve a wide variety of experimental goals in all common model systems, ranging from protein expression, localization, two-hybrid screening, and RNAi.Īll of these destination vectors include attR1 and attR2 and are therefore compatible with standard Gateway Entry clones, whether they are made by restriction enzyme cloning, BP clonase, or TOPO cloning. You may also use multisite Gateway cloning to assemble multiple fragments in a single destination vector. If your expression clone requires the transfer of only one fragment of interest into a destination vector, it is considered standard Gateway cloning. Variety of destinations an entry clone can be transferred to in gateway cloning. ► Learn more about restriction enzyme cloning Two small multicloning sites are located inside each attL site allowing standard restriction cloning reactions to be carried out. Restriction enzyme cloning into an Entry Vector, such as pENTR1A. You have a choice of three molecular approaches to create your Entry Clones.ġ. You can create your own Destination Vectors by adding a complete Gateway Cassette, which includes att sites and the selectable markers, into an expression vector of your choice. The ccdB gene and associated chloramphenicol gene are a universal feature of all Gateway vectors. The same ccdB gene provides negative selection against intact destination vectors. In this case, the plasmid is positively selected for with Ampicillin. The same pattern of positive and negative selection is used after performing LR clonase reactions to create your Expression clone. ![]() A ccdB gene, which is a suicide gene and will kill any bacteria that hosts itįollowing the BP reaction, which introduces your fragment of interest between the attP sites, all plasmids transformed will be able to survive Kanamycin selection, but only recombinants, which have lost the ccdB gene will be able to grow.A Kanamycin resistance gene which will positively select for the presence of the plasmid. ![]() Gateway cloning takes advantage of both positive and negative selection during the process of propagating vectors and selecting for recombinant plasmids.įor instance, the Donor Vector pDONR201 contains multiple genes for selection including: your fragment of interest is now located between attL sites, and ready for subsequent Gateway Reactions. The product you generate with your Entry Vector or Donor vector is an Entry Clone i.e. In contrast, Donor vectors require you to use PCR to add attB sites to your fragment of interest then use the Gateway BP Clonase reaction. An entry clone is a plasmid carrying a fragment of interest located between attL sites.Įntry vectors depend on conventional restriction enzyme cloning to introduce your fragment of interest between the attL sites.
0 Comments
Leave a Reply. |