The TOPO TA Cloning Kit® provides a highly efficient, 5-minute cloning strategy for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector. No ligase, post-PCR procedures, or PCR primers containing specific sequences are required. The plasmid vector is supplied linearized with single 3’ T overhangs for TA cloning and ‘activated’ (topoisomerase I covalently bound to the vector). PCR products must contain a single 3’ adenine overhang which allows the PCR inserts to ligate efficiently with the vector. If you use a high fidelity Taq (proofreading polymerase) that does not provide this overhang, a protocol for adding the overhang is included in the instruction manual. Reagents for this protocol are not included in the kit, however.
As a chemistry technician, I had no previous experience with sequencing. However, as our project developed it lead us to need quite a bit of sequencing work. Since I had access to a sequencer, I figured that I should learn how to do this to save time and money. After a little research, I decided to try this kit; from what I read, it couldn’t get any easier.
With a background in analytical chemistry, cloning was also foreign territory. Prior to this, I had never even streaked out a plate. My first few attempts at cloning were unsuccessful, but now it is working for me about 90% of the time. The pieces I am sequencing are very small DNA aptamers, about 60 bp. I find it helpful to add betaine to my PCR reactions to eliminate polymerase jumping during amplification. After PCR, I check the product on a gel. I do not do any sort of purification prior to cloning. I usually do at least 2 ligation reactions with 0.5 ul and 1 ul of neat PCR product. The instructions state that fresh PCR product must be used, but I often do my PCR in the afternoon and then do the ligation the following day with no problems.
I order the kit with TOP10 cells; as recommended in the protocol, I use 2 ul of the ligation reaction for transformation. I then streak out 50 ul of cells onto Xgal/IPTG LB Amp plates. The following morning, I always have well-spaced colonies, about 60-75% of which are white or light blue. I have found equal success in sequencing either white or light blue colonies, perhaps because my insert is so small.
The bottom line is, I would definitely recommend this kit, especially to someone who is new to sequencing.
Shelly D. Roper
Chemistry Technician
Conceptual Mindworks, Inc
Air Force Research Labs, HEPC