PCR amplification with a proofreading polymerase, like Pfu DNA polymerase, will leave you with a blunt end. However, another thermostable DNA polymerase, like Taq DNA Polymerase, adds a single nucleotide base to the 3’ end of the DNA fragment, usually an adenine, creating an “A” overhang. This “A” overhang can create difficulties when cloning the fragment is your end goal. You might consider creating a blunt end with Klenow or adding restriction sites to the ends of your PCR fragment by designing them in your primers. But why go through all those extra steps, when that “A” overhang allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors? Fewer steps? Who can argue with that?Continue reading “Cloning Blunt-Ended DNA Fragments is Hard: pGEM®-T Vectors Can Make It Easier.”
Q: Can PCR products generated with GoTaq DNA Polymerase be used to for T- vector cloning?
A: Yes. GoTaq® DNA Polymerase is a robust formulation of unmodified Taq Polymerase. GoTaq® DNA Polymerase lacks 3’ →5’ exonuclease activity and displays terminal transferase activity that adds a 3′ deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaq® DNA Polymerases (including the GoTaq® Flexi and GoTaq® G2 polymerases) will contain A-overhangs which makes them suitable for T-vector cloning with the pGEM®-T (Cat.# A3600), pGEM®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors.Continue reading “T-Vector Cloning: Questions, Answers and Tips”
Some thermostable DNA polymerases, including Taq, add a single nucleotide base extension to the 3′ end of amplified DNA fragments. These polymerases usually add an adenine, leaving an “A” overhang. There are several approaches to overcome the cloning difficulties presented by the presence of A overhangs on PCR products. One method involves treating the product with Klenow to create a blunt-ended fragment for subcloning. Another choice is to add restriction sites to the ends of your PCR fragments. You can do this by incorporating the desired restriction sites into the PCR primers. After amplification, the PCR product is digested and subcloned into the cloning vector. Take care when using this method, as not all restriction enzymes efficiently cleave at the ends of DNA fragments, and you may not be able to use every restriction enzyme you desire. There is some useful information about cutting with restriction sites close to the end of linear fragments in the Restriction Enzyme Resource Guide. Also, some restriction enzymes require extra bases outside the recognition site, adding further expense to the PCR primers as well as risk of priming to unrelated sequences in the genome.Continue reading “A Quick Method for A Tailing PCR Products”
A: The simplest way to clone PCR Products is to amplify the product using thermostable polymerases such as Taq, Tfl or Tth polymerase. These polymerases add a single deoxyadenosine to the 3´-end of the amplified products (3´-end overhang), and can be cloned directly into a linearized T-vector.
Q: What if my DNA polymerase has 3´ to 5´ exonuclease activity (i.e., proofreading activity) that removes the 3´-end overhang?
A: To clone PCR products that have been amplified with a polymerase that have proof reading activity into a T-vector, you will need to perform an A-tailing step using Taq DNA polymerase and dATP. Blunt ended restriction digest fragments can also be A-tailed using this method. The method below uses GoTaq Flexi DNA Polymerase (comes with a Mg-free reaction buffer), but any Taq DNA polymerase can be used.
Set up the following reaction in a thin-walled PCR tube:
1–4µl purified blunt-ended DNA fragment (from PCR or restriction enzyme digestion)
2µl of 5X GoTaq Reaction Buffer (Colorless or Green)
2µl of 1mM dATP (0.2mM final concentration)
1µl GoTaq Flexi DNA Polymerase (5u/µl)
0.6µl of 25mM MgCl2 (1.5mM final concentration)
Nuclease-free water to a final volume of 10µl
Incubate at 70°C for 15–30 minutes in a water bath or thermal cycler.
Q: What is a T-vector, and why are they used for cloning PCR products?
A: T vectors are linearized plasmids that have been treated to add T overhangs to match the A overhangs of the PCR product. PCR fragments that contain an A overhang can be directly ligated to these T-tailed plasmid vectors with no need for further enzymatic treatment other than the action of T4 DNA ligase.
For a complete PCR Cloning protocol, Visit the Cloning Chapter of the Promega Protocols and Applications Guide.