Ligate a very small insert into a vector - (Mar/14/2018 )

Hi. I want to ligate a small DNA fragment (a luminescence tag), 33bp into a vector 3.3kbp.

I do not know how. So I am wondering if I can design just one primer that covers that whole DNA fragment with RE sites included. Then I run PCR with sample (another vector that contains the tag). So double stranded DNA of the 33bp fragment should be produced. Then I will digest the purified PCR product with RE.

But instead of run gel and excise the band (because I do not think I can detect such small size of DNA in gel), can I just directly use the RE digested mixture and ligate to the linearized vector? And expect the 33bp fragment can ligate with the vector?

There are a couple of ways you can do this - As you say, you can either design primers and PCR the tag, then digest and clone. You may also be able to sub-clone the tag out of another vector using RE sites that are already there. The third method (and the one I would go with, as it is easy and quick for this length sequence) is to design two overlapping oligos, such that you have your desired sequence and restriction sites on each end (you can make them so that the sites are already overhanging if you think about it...), then anneal them and clone in. For this method you want relatively pure oligos, PAGE or HPLC will do, and you need to ensure it has the correct phosphorylations etc - though you can add/remove these yourself if you want to.

bob1 on Wed Mar 14 13:41:40 2018 said:

There are a couple of ways you can do this - As you say, you can either design primers and PCR the tag, then digest and clone. You may also be able to sub-clone the tag out of another vector using RE sites that are already there. The third method (and the one I would go with, as it is easy and quick for this length sequence) is to design two overlapping oligos, such that you have your desired sequence and restriction sites on each end (you can make them so that the sites are already overhanging if you think about it...), then anneal them and clone in. For this method you want relatively pure oligos, PAGE or HPLC will do, and you need to ensure it has the correct phosphorylations etc - though you can add/remove these yourself if you want to.

Thanks. Another vector with the tag does not have the RE sites that I want to ligate with the new vector.

I am considering your third method. So I will design the complementary primers with overhangs, but my question is I thought these lyphophilized primers/oligos purchased from outsource company should be pure enough. But such primers usually are dephosphorylated. Since the vector which I'll digest with RE is going to be phosphorylated, can I count on just the vector being phosphorylated and still be able to ligate with the annealed complementary oligos?

No - you need the insert phosphorylated. The vector can be phosphorylated or not, it doesn't matter other than that it might re-circularize if it is phosphorylated, though you can mostly avoid this by using non-compatible ends. You should always run a ligation control with just vector, just so that you have an idea of how much background re-circularization you have.

You can either buy phosphorylated oligos or phosphorylate them with T4 polynucleotide kinase.