Learning Module - Experiment 10B

Ligation of topA DNA into M13mp19.

In the last period we made and purified the blunt-ended PCR product. In this laboratory period, the fragment will be cloned into a phage vector, M13mp19.

In the first step, we will treat the vector with SmaI restriction enzyme and then remove the 5'-phosphates with Calf Intestinal Alkaline Phosphatase (CIAP). Why do we treat with CIAP?

Digestion with SmaI

In the second step, we combine the SmaI-digested, dephosphorylated vector with an excess of the purified PCR product. The mixture is incubated in the presence of T4 DNA ligase and ATP, and results in a covalently closed, double-stranded, recombinant M13mp19 molecule, which can be used to transfect a suitable E. coli host. Since this is not a directional cloning strategy, the insert can be ligated in either orientation.

Blunt end cloning

In the next laboratory period you will transfect XL-1 Blue cells with the product of today's ligation and select plaques for production of single-stranded DNA.