Researchers are always looking for products that help them save time, but they usually aren’t very happy about the price tags that sometimes come with these technologies. Fortunately, Promega has developed an exception to this rule with their pGEM-T Easy Vectors System. The pGEM-T products are linearized vectors with 5’T overhangs used for annealing the 3’A overhangs generated by Taq polymerase during PCR. After a standard PCR reaction, the products are quickly purified (either using spin columns or by phenol extraction (to remove the Taq polymerase)) and then ligated with the pGEM-T vectors. The ligation mix can then be transformed and blue/white screening can be used to identify positive transformants.
There are several things that I like about this product including (as mentioned above) its price, its ease of use and (most importantly) the success that I’ve had using it (price is irrelevant unless the product saves time and is successful). Using this system, PCR products can be ligated and transformed within 2 hours of amplification. The presence of inserts can be distinguished using blue white screening, and to screen insert size, one can use the 5’ and 3’ flanking Not I restriction sites. Since Not I is an eight base cutter, it is not frequently found within genomes. If your sequence of insert happens to have a Not I site there are many other unique flanking restriction sites to pick from. Finally, the vector is very reliable with a wide range of insert sizes. Over the last two years I have cloned inserts that range in size from 80 bp to 3.5 kb.
As always, the system is not perfect. Although this kit is fast, it is not the fastest kit on the market. There are other systems that do not require the removal of Taq polymerase, eliminating 30 minutes from the protocol (but these systems have a higher price tag).
Still, the pGem-T Easy Vector System is ideal if you are looking for an inexpensive TA cloning alternative. It’s an easy way to save time cloning PCR products.
Wayland Lim M.S.
Post-Graduate Researcher
UC-Davis