Plasmid construction and 3-point ligations

 
Don't forget to consider the trick of the 3-point ligation when it's not possible to find unique sites for certain restriction enzymes available in the lab...

The following example shows you how to get more cloning possibilities when using such a ligation.

Figure 1

Figure 1 shows a map of the plasmid, in which fragment 1 should be replaced by an A/B fragment from another plasmid (A and B are restriction enzymes).

Next figure 2, however, shows the problem..

Figure 2

There are 2 A sites and 3 B sites...

Figure 3

So, it will not be easy (two partial digestions..) to obtain a useful amount of fragment 2, for ligation to the new A/B fragment.
To avoid partial digestions (or an alternative strategy involving possibly expensive unique cutters), do the following:
search for a site in A2B2 which is unique in fragment 2.
In many cases, this will not be a problem

Imagine that you find such a site for enzyme C (see figure 4)

Figure 4

C1 is the unique C site in fragment 2. Any C sites outside fragment 2 (in this example C2) are not relevant.

Figure 5

So, the trick now is:
a 3-point ligation, using the following fragments:

  1. your new A/B fragment
  2. B/C fragment (purified from a B+C digest gel lane)
  3. A/C fragment (purified from an A+C digest gel lane)

You get the idea..

This works best, when A, B and C all generate sticky DNA ends.
Use standard ligase (0.2-1 unit), overnight at 16°C (do not use a Quick Ligation kit, or similar..).