![]() 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.
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