The Blunt-ended PCR Cloning Kit from GE Healthcare (formerly Amersham Biosciences) has everything that one would require for cloning any gene of interest. The kit provides a detailed step-by-step protocol and all the reagents, including competent cells and liquid media for their growth as well as the reagents necessary for carrying out the cloning reaction.
The cloning kit contains a pMOS Blue vector, which is a small (~2.8 kb), high copy number plasmid. All standard restriction sites are available in the multiple cloning site (MCS). The MCS is present within the lacZ gene fragment, which aids in simple blue/white selection of the clones through alpha complementation. An ampicillin resistance gene has also been incorporated for selection of bacterial cells which contain the plasmid. The vector is supplied ready to use: it is linearized via the EcoRV site and dephosphorylated. We have found very little self-ligation.
The cloning kit contains, in addition to the vector, competent cells (MOS Blue), SOC medium for growing transformed cells, T4 DNA ligase and all required buffers for carrying out the ligation reaction. A control insert is also provided for testing the efficiency of the reaction and the supplied components. The material in the kit is sufficient for 40 cloning reactions. In our laboratory, we do not use the competent cells that are supplied along with the kit, as we have found these cells not to work efficiently enough. Upon transformation of the supplied MOS Blue cells with the ligation reaction following the protocol in the instruction booklet, I obtained very few colonies (~15-20). However, I could still find the positive clones in them with the help of blue-white selection. But for our routine cloning experiments, we prepare our own competent cells. We believe that the competent cells provided along with the cloning kit lose their efficiency during shipment, although they are well-packed in dry ice.
If dephosphorylated primers have been used in PCR, the PCR product must be phosphorylated prior to its ligation with the pMOS Blue vector. For such cases, kinase enzyme and buffers are also available in the kit. Addition of ATP is not required for the phosphorylation reaction, as the buffer includes optimal amounts of ATP.
In the experiments carried out in our laboratory, we have obtained over 95% positive clones in most cases. We have tried cloning up to a 3.2 kb gene with a success rate of over 70%. Smaller fragments of about 550 bp have given us nearly 95% positives. Moreover, if the protocol is followed properly, then the entire reaction can be finished in just one hour.
Due to the presence of almost every standard restriction site in the MCS, care should be taken when cloning genes, to check for the presence of restriction sites that are present in both the gene and the MCS.
The major benefit of using the Blunt-ended PCR Cloning Kit over buying the reagents necessary for cloning individually is in its cost effectiveness. Additionally, the pMOS Blue vector, owing to its high copy number and easy selection of positive colonies, offers a rapid cloning system.