What is pENTR d TOPO?
Additional Information. The pENTR™/D-TOPO® Cloning Kits utilize a highly efficient, 5-minute cloning strategy (“TOPO® Cloning”) to directionally clone a blunt-end PCR product into a vector for entry into the Gateway® System or the MultiSite Gateway® System.
What is directional TOPO cloning?
Directional TOPO cloning enables cloning of blunt-ended PCR products in a 5´→3´ orientation directly into a expression vector using a 5-minute ligation reaction, thereby eliminating subcloning steps and saving you time. Topoisomerase I then ligates the PCR product in the correct orientation.
What is pENTR?
The pENTR™ vectors allow restriction cloning of the gene of. interest for entry into the Gateway™ system available from Invitrogen. A choice of pENTR™ vectors is available (see table below) for optimal expression of your gene of interest after recombination with the Gateway™ destination vector of choice.
How does Gateway cloning work?
You can use Gateway cloning to insert multiple DNA fragments into many vectors at once in the same tube. You can clone up to 4 DNA fragments, in a specific order and orientation, in one tube, into one Gateway vector to produce the desired expression clone. This is possible thanks to the Gateway vectors’ design.
Is topoisomerase a restriction endonuclease?
The key to TOPO cloning is the enzyme DNA topoisomerase I, which functions both as a restriction enzyme and as a ligase. Its biological role is to cleave and rejoin DNA during replication.
Is DNA ligase used in PCR?
The equivalent of DNA polymerase I and DNA ligase are also unnecessary due to the absence of RNA primers and Okazaki fragments during the process of PCR.
What is a gateway vector?
Gateway vectors contain modified versions of the att sites so that scientists can easily clone in their desired DNA sequences. Gateway technology relies on the two reactions described below: The BP Reaction takes place between the attB sites flanking the insert and the attP sites of the donor vector.
What are gateway cloning vectors?
Gateway® Cloning is a universal cloning technique developed by Invitrogen life technologies. Gateway® Cloning Technique allows transfer of DNA fragments between different cloning vectors while maintaining the reading frame. Using Gateway®, one can clone/sub clone DNA segments for functional analysis.
How does the Pentr™/D-TOPO® vector work?
The pENTR™/D-TOPO® vector (Figure 1) includes M13 and T7 primer sequencing sites and attL recombination sites flanking the PCR product insertion site. This allows clones to be easily sequence verified and recombined into your choice of attR containing Gateway® destination vectors.
How do the Pentr™/D-TOPO® Cloning kits work?
The pENTR™/D-TOPO® Cloning Kits utilize a highly efficient, 5-minute cloning strategy (“TOPO® Cloning”) to directionally clone a blunt-end PCR product into a vector for entry into the Gateway® System or the MultiSite Gateway® System.
What is vectorvector database?
Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. This is a free resource for the scientific community that is compiled by Addgene. This page is informational only – this vector is NOT available from Addgene – please contact the manufacturer for further details.
What is the pcr8/gw/TOPO vector used for?
The pCR8/GW/TOPO vector enables efficient TOPO-TA cloning. These vectors easily facilitate multipurpose use: rapid recombination into a variety of Gateway Destination vectors, convenient sequencing, robust selection in E. coli with spectinomycin resistance, and easy exicision of insert DNA with flanking EcoR I sites ( Figure 3).