Gateway and TOPO Cloning

Invitrogen market a number of kits and cloning vectors designed to simplify the cloning and subcloning of DNA fragments such as those generated by PCR amplification. Amongst the most popular are the Gateway and TOPO kits. For detailed information on the concepts, availability and pricing of these kits, please visit the Invitrogen web site. MacVector includes support for simulating Gateway and TOPO cloning manipulations so that you can accurately document the sequences of the constructed molecules using a simple point and click interface.

Vectors

MacVector includes a variety of Invitrogen vectors nicely formatted for use in Gateway, TOPO-TA and Zero Blunt cloning manipulations. You can download additional vectors for the Invitrogen web site or from many other places, including the NCBI. If the vectors you download do not have features or don't have nice formatting like the one's supplied with MacVector, you can use the auto-annotation function of MacVector to quickly annotate the sequences based on the vectors in the /MacVector/Common Vectors/Invitrogen/ folder.

TOPO TA and Zero Blunt Cloning

MacVector lets you replicate a TOPO TA or Zero Blunt cloning using the following simple steps;

1. Ensure that the Common Enzymes RE file is selected in the Automtic RE Analysis preferences. Also ensure that either ALL ENZYMES are being displayed or that the TopoTA/BLNT cloning site is selected if not.
2. Select the region in the Map View using the Select Sequence tool in a source DNA molecule corresponding to the PCR fragment.
3. Choose EDIT | DIGEST to place the blunt ended fragment on the Cloning Clipboard.
4. Open the target vector molecule and select the TopoTA/BLNT site.
5. Drag the fragment from the Cloning Clipboard to the vector to insert the fragment into the vector.

Gateway Cloning

MacVector automatically displays the att sites used in Gateway cloning in the graphical Map display of a sequence. As with TOPO TA cloning, replicating Gateway cloning just requires a few simple steps;

1. To replicate a Gateway cloning, first select a pair of att sites in your source vector, as shown at the end of the Topo cloning example above
2. Click DIGEST. The fragment will appear in the Cloning Clipboard.
3. Select the fragment in the Cloning Clipboard.
4. Open the destination vector. Any compatible sites will be highlighted. Click on the corresponding target att sites
5. Drag the fragment from the Cloning Clipboard to the vector to insert the fragment into the vector.

Because the attL1/attR1 and attL2/attR2 sites have a slightly different core sequence, the source fragment can only recombine into the target sequence in one orientation. MacVector automatically flips the source if necessary so that it always gets inserted in the biologically correct orientation.

Related Topics.

Click Cloning

Cloning Clipboard

Performing Click Cloning

End modification

Restriction enzyme

Automatic Restriction Enzyme Analysis