The PCR-TRAP Cloning System is an extremely efficient way to clone very small amounts of PCR product. This system uses a third generation cloning vector that features a positive-selection for DNA inserts that involves the phage Lambda repressor gene, cI. When cI is transcribed, it codes for a repressor protein that binds to the Lambda right operators Or1 to Or3 of the cro gene. This turns off the promoter that drives the TetR gene on the plasmid. Cloning of the PCR product into the cI gene leads to the inactivation of the repressor gene and thus the expression of the TetR gene. Because a significant fraction of PCR products do not contain 3' overhanging A’s, there is no need for any post-PCR manipulation before cloning. The cloned PCR products can be quickly and easily retrieved by colony-PCR to confirm the presence of an insert or probes can be generated using primers that flank the cloning site (provided in the kit). If cDNA amplified by an RNAimage Kit (GenHunter) is cloned into a PCR-TRAP System, the insert, in theory, can also be excised by HindIII digestion. The flanking primers also allow the cloned PCR product to be readily sequenced. The kit also contains GH Competent cells as well as a positive control, the lacZ gene, which, after PCR and cloning, will confer blue color upon X-gal staining.
The protocol and the system are very simple and efficient. The insert cloning is almost error-free and the user doesn't even need to calculate the molecular ratio between vector and insert to get your PCR product in. Unfortunately the vector that comes with this system does have 2 problems. The first is that the sequence is not provided to the customer because it is still patent pending. This can make working with the vector difficult, since it is important to know all of the restriction sites present in a vector. The second issue is that the vector contains three HindIII sites as do the random primers provided with the RNAimage kit (which is primarily what I use this system with). This means that it is often impossible to excise the insert by HindIII digestion because the bands coming from the vector are often overlapping the insert bands. In these cases, the only way to get the insert out of the PCR-TRAP vector is PCR amplification and subcloning in another plasmid.
Despite these issues, I would have to say that the PCR-TRAP Kit is an excellent system for PCR cloning because it can efficiently clone really small amount of PCR product. It assures you an excellent backup system for precious and often unknown genes.
Luca Carta, Ph.D.
Mount Sinai School of Medicine
New York, New York