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.
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?
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.
Before you begin your subcloning, you need to know: The restriction enzyme (RE) sites available for subcloning in your parent vector multiple cloning region (or in the insert if you need to digest the insert); the RE sites available in the destination vector multiple cloning region (MCR); and if these same sites also occur in your insert. Once you know this information, you can use the chart below to decide which subcloning strategy to use.
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®).
Escherichia coli remains the first choice of many researchers for producing recombinant protein for functional studies due to its ease of use, well established protocols, rapid cell growth and low cost of culturing. Researchers often need to clone using an E. coli host with good transformation characteristics first, then transfer the desired clone to the expression host. We have developed a new E. coli host KRX, that provides protein yields comparable to those of BL21(DE3) but with much higher transformation efficiencies. Continue reading “Efficient Cloning and Expression of High Protein Yields Using KRX Cells”
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