Welcome to the Enzyme Free Cloning (EFC) introduction page. Since we perform a lot of high throughput (HTP) cloning in our laboratory, we set up a strategy to clone PCR products without any enzymatic treatments after the PCR (de Jong et al., 2007). This eliminates essentially all variability during cloning on the side of the insert, which means that if we see a decent PCR product, we virtually always have the expression vector next day. In brief: we create 14 bp long single stranded overhangs in the vector using T4 DNA polymerase, and we create complementary 14 bp long overhangs in the PCR product insert using a PCR trick called hetero-stagger PCR. These overhangs are so long, that simply mixing the two will be enough to create a vector: the bacterial DNA repair machinery will do the rest for you. We use EFC as a combination of two previously described cloning tricks: Ligation Independent Cloning (Aslanidis and de Jong, 1990) and Hetero-Stagger PCR cloning (Liu, 1996, Zeng, 1998, Shih et al., 2002). Previously reported was a version in which both insert and vector were amplified using hetero-stagger PCR cloning (Tillett and Neilan, 1999). Since HTP cloning consumes a lot of vector, and amplification of entire vectors is not very efficient, we have found the combination of LIC for the vector and EFC for the PCR products to be most robust (de Jong et al., 2007). Like with any cloning strategy, the insert that needs to be transferred to a vector of interest should contain terminal sequences that are compatible with the entry site in the vector. For preparation of the vector, we use a technique called Ligation Independent Cloning (Aslanidis and de Jong, 1990). We open up the vector using a restriction enzyme – SacII, in our vectors – and generate 14 bp long cohesive ends using T4 DNA polymerase treatment in the presence of a single nucleotide. While the 3′->,5′ exonuclease activity of the polymerase degrades the 3′ terminus, the 5′->,3′ DNA polymerase actvity starts building in nucleotides as soon as it can. Therefore, the following vector termini, generated after SacII treatment of our vectors: To create 14 bp sticky overhangs in the insert, we perform two PCR reactions for each insert following the hetero-stagger PCR technique (Liu, 1996) as described in the figure: After purification of the two PCR products, we hybridize the products, generating complementary cohesive ends in 25% of all hybridization events. Then, we add the vector, incubate for 5′ at room temperature and transform bacteria. There is no need for gel purification of the PCR product, but we do use a magnetic bead purification to get rid of the polymerase and the primers, which can cause false positives (colonies, no insert). This can be done on a biorobot. Well, any LIC compatible vector works, but if you decide to use our suite of bacterial expression vectors, you need to order the following primers for your genefragment of interest: We are expanding our suite with eukaryotic expression vectors and hope to update the relevant pages as we go along. If you use our vectors or strategy, we suggest you cite our paper describing both (de Jong et al., 2007). Good luck and happy cloning! Source.