The StrataClone™ PCR Cloning Kit is designed for efficient cloning of PCR fragments. The kit contains StrataClone™ Vector Mix, StrataClone™ Cloning Buffer, SoloPack® PCR Cloning Competent Cells, StrataClone™ Control Insert and pUC18 Control Plasmid. This kit utilizes the 3’-adenosine overhangs produced during amplification with
Taq. Efficient ligation occurs with vector arms charged with topoisomerase I on one end and a lox P recognition sequence on the other end. The PCR fragment and vector come together through A-U base pairing. Following base pairing, topoisomerase I mediates strand ligation. The resulting ligated molecule is then transformed into a competent cell line that transiently expresses Cre recombinase followed by Cre-mediated recombination between the vector loxP sites and resulting in circular DNA molecule pSC-A.
We purchased the StrataClone™ PCR Cloning Kit after many unsuccessful attempts to either directly sequence PCR products or clone then sequence PCR fragments. We performed PCR on cDNA derived from reverse transcription of total RNA isolated from retina. The aim was to sequence multiple forms of p107 protein that were produced in normal human retina and in retinal tumors (retinoblastoma). Since multiple forms of cDNA were produced, the amount of PCR product generated from each cDNA was not enough for direct sequencing. Cloning by the classical approach was inefficient due to the low ligation efficiency of our blunt-ended PCR products.
Our very first use of the StrataClone™ PCR Cloning Kit proved to be successful: We got PCR product from all forms of cDNA ligated into pSC-A in one ligation (“shotgun” approach). The reaction was extremely efficient. We screened only 24 colonies and 80% of them had one of the fragments ligated into pSC-A. Ampicillin resistance and blue-white screening made the selection of colonies for miniprep simple. Screening the minipreps for insert was an easy task since pSC-A polylinker contains many restriction enzyme sites. Sequencing was also trivial with T3 and T7 primers; there was no need to design additional internal primers. The materials in the kit were provided in generous portions: Truly enough to do 10 reactions, each using a separate aliquot of competent cells (provided). The instruction manual was very easy to follow and included instructions for sample prep, clone screening and sequencing.
Since mutational analysis requires the use of proofreading DNA polymerases which do not produce 3’-A overhangs it is crucial to remember to use a protocol that adds 3’-A overhangs after PCR amplification. This protocol is very straightforward and it requires use of Taq polymerase. Overall, we find the StrataClone™ PCR Cloning Kit highly efficient for cloning of multiple PCR products derived from a single reverse transcription reaction. Measured in efficiency, time consideration and simplicity, this product by far justifies the cost of kit per sample.
Sanja Pajovic, PhD
Research Associate
Ontario Cancer Institute
Applied and Molecular Oncology