Cloning trick: ligation of multiple inserts

[2013.02.26 Edit: A number of people are finding this through Google searches. I don’t have an updated post on the topic, but if you’re trying to assemble multiple DNA fragments then I suggest looking into Gibson Assembly. NEB sells* a dead-simple mastermix, which is a bit pricey per reaction (I just make my reactions half the size) but comes out to cheap when you take into account the cost of labor (so long as your PI values your time…).]

I’ve spent the last couple months building a plasmid library, and in the process I thought of a trick. Ligations, perhaps the worst part of cloning, are notoriously finicky reactions. The goal is to take several pieces of linear DNA, where the ends of the pieces can only connect in a certain way, and then use an enzyme (T4 Ligase) to sew them all together into one piece (in my case, a circular plasmid).

Figure 1. Ligase (2HVQ.pdb) rendered in PyMOL. Click to see a crappy animated GIF!

I needed to insert three fragments at once into a single backbone. In my ignorance (from my lack of experience) I thought ligating four fragments should work just as well as two, so I just threw them all together and ran the reaction. The result was a mess, and when I tested 40 different clones afterwards not a single one was correct. So I started adding them one piece at a time which, obviously, was going to take three times as long.

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