StrataClone™ Blunt PCR Cloning Kit From Stratagene

Cloning of PCR products is important for most, if not all, researchers working in the life sciences. Traditionally, cloning was achieved by generation of a PCR fragment using primers designated to add restriction site overhangs at the 5’ and 3’ ends. This amplified DNA fragment was subsequently ligated into a plasmid that must contain identical restriction sites to the restriction sites overhangs on the PCR fragment; the plasmid, of course, had to be digested prior to ligation to make these overhangs available to the PCR product. In addition, these sites must be unique in the plasmid. Furthermore, traditional cloning could be tricky as a number of restriction enzymes have minimum length requirements in order to efficiently digest the DNA; thus, primers sometimes had to contain extension sequences. This requirement creates difficulty with primer design as the primers tend to be long, more expensive, and more difficult to design with regard to Tm, GC content, hairpins, and undesired repeats. Buffer compatibility for restriction enzymes may also cause difficulties. Although very reliable and well characterized, this method requires laborious work and could take a long time to complete.

Stratagene put an end to these limitations when they came out with a very efficient cloning kit: The StrataClone™ Blunt PCR Cloning Kit (Cat No. 240207 for 20 reactions or 240208 for 10 reactions). They have other similar kits and after visiting their website, I chose this one. The kit is compatible with any DNA template, be it genomic DNA, cDNA or a vector/plasmid. There is no need to purify the amplified PCR product from the PCR reaction mix and once the PCR product is generated, the procedure is simple and easy to complete. There is no need to pre-digest the plasmid and the enzymatic ligation reaction takes 5 minutes to complete.

So how does it work? In brief, the StrataClone™ blunt PCR cloning technology utilizes two major enzymatic activities of topoisomerase I from Vaccinia virus and Cre recombinase from bacteriophage P1. Topoisomerase I cleaves the DNA strand downstream of the sequence 5’ –CCCTT at the phosphodiester backbone, creating a DNA-enzyme covalent high energy intermediate. This high energy intermediate is used later for subsequent religation to heterologous DNA molecule (the PCR product). The Cre recombinase enzyme resides in the competent cells supplied with the kit and catalyses the recombination between two loxP recognition sequences resulting in a circular plasmid.

The cloning vector mix contains two pieces of blunt-end linear vector DNA. One piece contains at the 5’-end the loxP recognition sequence and the 3’-end is covalently charged with the topoisomerase I. The second piece contains at the 5’-end a covalently charged topoisomerase I and the 3’-end contains the loxP recognition sequence. Prior to ligation, the PCR product must be generated. DNA polymerases that add overhanging nucleotide(s), such as Taq polymerase which adds extra adenines, are not suitable because the topisomerase I will not recognize it. In such cases, the PCR product should be treated with an appropriate exonuclease.

By using a proofreading polymerase such as Pfu, a blunt-ended PCR product is then efficiently ligated by topoisomerase I-mediated strand ligation to the linear vector DNA pieces. Typical ligation takes 5 minutes to complete and efficient ligation resulting with a linear dsDNA where the PCR product is sandwiched between the two vector parts. The resulting linear DNA contains the following: 1) pUC ori, 2) P_LAC, 3) two lacZ’ separated by the ligated PCR fragment, 4) MCS, 5) ampicillin cassette, 6) two loxP sequences as detailed above. This DNA molecule is then transferred to the competent cells, StrataClone™ SoloPack®, which transiently express Cre recombinase, in order to circularize the linear DNA molecule produced by the topoisomerase I-mediated ligation. Transformation is performed via heat shock at 42°C for 45 seconds and is highly efficient. The cells are chemically competent and are supplied in a single tube format (200 µL/aliquot). The strain contains the lacZM15 mutation and therefore, supports screening by blue-white selection. The lacZ is constitutively expressed so there is no need to induce expression with IPTG. White colonies indicate cells transformed with plasmid pSC-B harboring the PCR product and blue colonies indicate cells transformed with a self ligated pSC-B missing the PCR fragment.

A good and fast alternative to blue-white screening is to perform a colony PCR test and verify the presence of the PCR fragment in the plasmid by DNA gel electrophoresis. The primers that were used for generating the PCR product can be used here and since high fidelity and overhangs are not issues for this test, Taq polymerase is recommended.

To conclude, this cloning kit is highly recommended. Traditionally, I devoted two to four weeks for cloning using the traditional method. Using this kit, I completed synthesis of a blunt-end 0.95 kb PCR product, cloning and confirmation of positive clones by colony PCR in four days. (Sequencing confirmation took additional two days.) Equally fast, a coworker cloned an 82 bp DNA PCR product, indicating that the size of insert is not likely to affect your success and time schedule. The handbook supplied with the kit is simple and easy to follow. It is important to remember that orientation of the insert can be verified only by sequencing, but since the efficiency is high (~500 colonies/100 µL transformants) and positive clones are predominant, this is not a big deal. I highly recommend this kit as a very easy and useful for general cloning of PCR products.