I was blasting a holiday music playlist while driving recently, and Presley’s Blue Christmas played. I couldn’t get the phrase “Christmas Cloning Blues” out of my mind, and by the time I arrived at my destination, this happened:
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?
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.
Restriction enzymes recognize short DNA sequences and cleave double-stranded DNA at specific sites within or adjacent to these sequences. These enzymes are the workhorse in many molecular biology applications such as cloning, RFLP, methylation-specific restriction enzyme analysis of DNA, etc. Restriction enzymes with enhanced capabilities can help you streamline and shorten these workflows and improve success of restriction enzyme digestion.
A subset of Promega restriction enzymes offer capabilities that include rapid digestion of DNA in 15 minutes or less, ability to completely digest DNA directly in the GoTaq® Green Master Mix, and Blue/White Cloning Qualification which allows for rapid, reliable detection of transformants.
To learn more about restriction enzymes and applications, check out Restriction Enzyme Resource on the web. The resource provides everything from information on restriction enzyme biology to practical information on how to set up and design a restriction enzyme digestion. This resource also contains useful online tools, including the Restriction Enzyme Tool, to help you use enzymes more effectively. It helps you choose the best reaction buffer for double digests, find the commercially available enzyme that cuts your sequence of interest, find compatible ends, and search for specific information on cut site, overhang isoschizomers and neoschizomers by enzyme name.
For added convenience, you can download the mobile app available for iOS devices and use the Restriction Enzyme Tool to plan your next digest.
For additional information regarding Restriction Enzyme Digest, reference the supplementary video below.
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.
Ah, the wonders and frustrations of cloning. We’ve all been there. After careful planning, you have created the cloned plasmid containing your DNA sequence of interest, transformed it into bacterial cells and carefully spread those cells on a plate to grow. Now you stand at your bench gazing down at your master piece: a plate full of tiny bacterial colonies. Somewhere inside those cells is your DNA sequence, happily replicating with its plasmid host. But wait – logic tells you that not ALL of those colonies can contain your plasmid. There must be hundreds of colonies. Which ones have your plasmid? You begin to panic. Visions of yourself old and grey and still screening colonies flash through your mind. At the next bench, your lab-mate is cheerfully selecting colonies to screen. Although there are hundreds of colonies on her plate as well, some are white and some are blue. She is only picking the white colonies. What does she know that you don’t?
Tailing blunt-ended DNA fragments with TaqDNA Polymerase allows efficient cloning of these fragments into T-Vectors such as the pGEM®-T Vectors. This method also eliminates some of the requirements of conventional blunt-end cloning — Fewer steps, who can argue with that?
Cloning is a fickle process that can make even the most seasoned bench scientists scream in frustration. By the time you perform a colony PCR and run the gel to check for your insert, you’ve invested several days in preparing these transformed cells. But then, the unthinkable happens. When you image your gel…the target band is missing.
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 (proof reading) and also displays non-template–dependent terminal transferase activity that adds a 3′ deoxyadenosine (dA) to product ends. As a result, PCR products amplified using GoTaq® DNA Polymerase will contain A-overhangs which makes it suitable for T-vector cloning.
We have successfully cloned PCR products generated using GoTaq® and GoTaq® Flexi DNA Polymerases into the pGEM®-T (Cat.# A3600), pGEM®-T Easy (Cat.# A1360) and pTARGET™ (Cat.# A1410) Vectors.
Q: Can GoTaq® Long PCR Master Mix be used for T-Vector Cloning?
A: Yes it can. GoTaq® Long PCR Master Mix utilizes recombinant Taq DNA polymerase as well as a small amount of a recombinant proofreading DNA polymerase. This 3´→5´ exonuclease activity (proof reading) enables amplification of long targets. Despite the presence of a small amount of 3´→5´ exonuclease activity, the GoTaq® Long PCR Master Mix generates PCR products that can be successfully ligated into the pGEM®-T Easy Vector System.
We have demonstrated that GoTaq® Long PCR Master Mix successfully generated DNA fragments that could be ligated into pGEM®-T Easy Vector System without an A-tailing procedure, and with ligation efficiencies similar to those observed with the GoTaq® Green Master Mix.
While T-vector cloning is commonly used for PCR-amplified inserts, restriction enzymes still have their uses. For example, you can ensure directional cloning if you digest a vector with the same two enzymes like BamHI and EcoRI that are used to digest your insert. This way, the insert can only be cloned in one direction. However, there are other cloning techniques that can be used to modify the end of vectors and inserts after restriction enzyme digestion and prior to ligation. Continue reading “Cloning Tips for Restriction Enzyme-Digested Vectors and Inserts”
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