Working with homology based cloning

This section gives a quick overview of the Homology Based Cloning wizard. For a full description, see Detailed description of the Homology Based Cloning wizard.

General options

General options are at the top of the wizard. These include the position of the insertion site in the vector, the maximum primer and overhang lengths as well as option to set the Tm and overhang length for all primers at once. There is also a diagram of the vector including the inserts, where each sequence has a different colour (figure 23.29).

Image cloning_wizard_top
Figure 23.29: The top section of the wizard contains general options.


Each sequence is displayed individually, with a coloured bar to the left and a vertical scroll bar at the bottom. The top sequence is the vector, with the insert sequences displayed further down.

The order of the sequences reflects how they will be assembled into the vector, and the overhangs on the primers support this assembly order.

Vector, inserts, primers and overhangs are color coded (figure 23.30):

For each sequence, you can adjust primer and overhang lengths and add bases between primers and overhangs.

The vector sequence is considered circular and primers are depicted as pointing away from each other in order to amplify the circular sequence. Inserts are considered linear, and primers are placed at the ends of the insert sequence pointing towards each other in order to amplify the linear sequence (figure 23.30).

Image cloning_wizard_sequences
Figure 23.30: Top: The vector sequence and primers with overhangs. The grey sequence between the primers is not included in the PCR product. Bottom: An insert sequence and primers with overhangs.