One of the easiest methods for cloning blunt-ended DNA fragments including PCR products is T-vector cloning, such as with pGEM®-T or pGEM®-T Easy Vector Systems. This method takes advantage of the “A” overhang added by a PCR enzyme like Taq DNA Polymerase. T vectors are linearized plasmids that have been treated to add 3′ T overhangs to match the A overhangs of the insert. The insert is directly ligated to the T-tailed plasmid vector with T4 DNA ligase. The insert can then be easily transferred from the T vector to other plasmids using the restriction sites present in the multiple cloning region of the T vector.
Proofreading polymerases like Pfu do not add “A” overhangs so PCR products generated with these polymerases are blunt-ended. In a previous blog, we discussed a simple method for adding an A-tail to any blunt-ended DNA fragment to enable T-vector cloning. Below, we think about the next step: Ligation.
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. In order to streamline and shorten these workflows, restrictions enzymes with enhanced capabilities are desirable.
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 use restriction enzymes. This resource also contains useful online tools 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 and use the Restriction Enzyme Tool to plan your next digest.
For additional information regarding Restriction Enzyme Digest, reference the supplementary video below.
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? Continue reading “Selecting the Right Colony: The Answer is There in Blue and White”
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.
Let’s face it, most lab techs and purchasing agents aren’t all that happy when you send them an Instagram picture of your latest lunchroom-napkin cloning strategy as your order form for your next big cloning experiment. So we have created the CloneWeaver® Workflow Builder. You can transfer your brilliance easily from that lunchroom napkin to an orderly email or print out of every vector, enzyme, purification kit, and transfection reagent your next big molecular cloning experiment requires. You can even save your one-of-a-kind “cloning kit” for future endeavors.
The CloneWeaver® tool will walk you through every step of the molecular cloning process from selecting a vector to finding a transfection reagent for mammalian cells. So if you are starting a new project, we are with you every step of the way. We will help you find restriction enzymes and even remind you about markers and biochemicals that you may want to have on hand for your experiment. Within the tool we have links to additional resources like our RE Tool and catalog pages if you need more help.
Already have a favorite vector and a freezer full of restriction enzymes? No problem, skip those steps and move on to getting the perfectly sized nucleic acid markers or the particular polymerase your experiment requires.
Are you teaching a molecular genetics course? CloneWeaver® workflow builder is perfect for creating the list of laboratory reagents you are going to need for your students—and you will have this same list as a starting point for other lab experiments or classes later on because you can save the lists that you build. You can even pass them along to other professors.
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”
You have PCR amplified your insert of interest, made sure the PCR product is A tailed and are ready to clone into a T vector (e.g., pGEM®-T Easy Vector). The next step is as simple as mixing a few microliters of your purified product with the cloning vector in the presence of DNA ligase, buffer and ATP, right? In fact, you may need to consider the molar ratio of T vector to insert.
When I was a graduate student, I often found myself doing directional cloning to engineer the perfect construct. Because this work occurred during an era when all graduate students had to walk uphill through the snow to the lab everyday (even in Atlanta, GA), my cloning strategy planning sessions usually went something like this:
Approach the only laboratory PC and coax a Dos-based sequence analysis program to perform a restriction enzyme analysis on my DNA sequence of interest. Then I would print this analysis out on the dot-matrix printer, which was usually out of paper. Next I would search through the lab files of technical information for the photocopy of the map of the desired vector, sit at lab bench with the all of the information, and select several enzymes from each end of the desired insert, which I would compare to the available enzymes in the Multiple Cloning Region of the vector.
Usually, I would discover that using these enzymes, I could only put my insert into the vector in the wrong orientation. So I would look at the information again and find another pair of enzymes. Eventually I would search through piles of biotech catalogs to see what enzymes were actually commercially available, compatible and would result in the desired construct. After much wailing and gnashing of teeth, I would go to the freezer only to find that someone had used all of the vector I prepped.
Fortunately many of the headaches of designing double digests are now distant memories thanks to a really handy restriction enzyme digest tool available on the Promega mobile application (available for iPhone® and iPad®).