Strataclone PCR Cloning Kit from Agilent Technologies

By now, cloning procedures are routinely carried out in biology laboratories. Although their widespread utilization has prompted companies to produce a plethora of kits, a good, high quality cloning kit is not so easy to find. The Strataclone PCR Cloning Kit seems to be one of those, fulfilling almost all of the researcher’s needs. It is a robust kit with a relatively easy procedure, which permits the cloning of short and large PCR fragments in just two hours.

Its technology uses the activities of Vaccinia virus topoisomerase I, which forms a covalent DNA–enzyme intermediate, and Cre recombinase, from bacteriophage P1, which catalyzes recombination between two loxP recognition sequences. Topoisomerase I and loxP recognition sequences flank each arm end of the PCR cloning vector. PCR products are efficiently ligated (within 5 minutes) by topoisomerase I; the resulting linear molecule is then transformed into a competent (and dedicated) cell line engineered to transiently express Cre recombinase enzyme. Cre-mediated recombination between the vector loxP sites creates the circular DNA molecule which includes a lacZ´ alpha-complementation cassette for blue-white screening. It is noteworthy that it is not necessary to induce lacZ´ expression with IPTG when performing blue-white screening with the provided competent cells. It has to be taken into account that if on the one hand, the use of dedicated cells helps to speed the procedure, but on the other hand, it limits the flexibility of the kit and slightly increases its cost.

The addition of an adenine residue to the 3´-end of both strands of the PCR product is needed, hence the manufacturer suggests the use of indicated high-fidelity PCR enzymes, but don’t worry: if you have your personal PCR enzyme for cloning applications, it can work fine. An additional short protocol to add 3´-A overhangs is provided in the kit, though I have never used this protocol.

The PCR product should be <3kb to get the best results, although the company declares that the kit has been used to clone PCR products up to 9.2 kb in length. I routinely amplify and clone fragments of 500-1500 bp. (If multiple PCR products are observed on the gel, a gel purification procedure can be performed.)

The ligation reaction is prepared by combining StrataClone Cloning Buffer, PCR product or StrataClone Control Insert, and StrataClone Vector Mix and incubating just 5 minutes at room temperature. The transformation of StrataClone SoloPack competent cells is similar to that of other strains (add ligation reaction to cells, incubate on ice 20 minutes, heat-shock at 42°C for 45 seconds and incubate on ice 2 minutes). Pre-warmed LB medium is added and cells are incubated at least 1 hour at 37°C with agitation. Finally, after plating the transformation mixture on 2% X-gal LB-antibiotic plates (IPTG is not necessary with this strain), incubation of plates overnight at 37°C is carried out. Miniprep DNA from selected white or light blue colonies using standard protocols and then you have your cloned DNA in few hours.

In conclusion, this cloning kit is highly recommended for its time savings, its double antibiotic site for cell antibiotic resistance (amp/kan) and the simple and easy to follow handbook.