From Napkin Sketch to “Custom Kit”: CloneWeaver® Workflow Builder Gets Your Cloning Organized

20161018_150403Let’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.

clone_weaverAlready 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.

So, if molecular cloning is in your future, let us help you get organized. Try the CloneWeaver® Workflow Builder.

Cloning Tips for Restriction Enzyme-Digested Vectors and Inserts

Cartoon created and owned by Ed Himelblau

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

Successful Ligation and Cloning of Your Insert

Ligation and cloningYou 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.

After the insert DNA is prepared for ligation, estimate the concentration by comparing the staining intensity of your PCR product with that of DNA molecular weight standard of similar size and known concentrations on an ethidium bromide-stained agarose gel. If the vector DNA concentration is unknown, estimate the vector concentration by the same method. Test various vector:insert DNA ratios to determine the optimal ratio for a particular vector and insert. In most cases, a 1:1 or 1:3 molar ratio of vector:insert works well, but you may want to consider 1:5, 5:1 and even a 10:1 ratio. The following example illustrates the calculation of the amount of insert required at a specific molar ratio of vector:insert.

[(ng of vector × kb size of insert) ÷ kb size of vector] × (molar amount of insert ÷ molar amount of vector) = ng of insert

Example:
How much 500bp insert DNA needs to be added to 100ng of 3.0kb vector in a ligation reaction for a desired vector:insert ratio of 1:3?
[(100ng vector × 0.5kb insert) ÷ 3.0kb vector] × (3 ÷ 1) = 50ng insert

Our BioMath Calculator is an easy way to calculate the molar ratio of vector to insert for ligation.

The vector:insert ratio changes, depending on the insert, even if you use the same vector. If you use the same vector:insert ratio for many different inserts and the insert size increases or decreases, recalculate the amount of insert needed for ligation using the equation above or our handy BioMath Calculator to ensure the molar ratio stays the same.

For more information on cloning, consult the Cloning chapter of the Protocols and Applications Guide.

Restriction Enzyme Digestion: Capabilities and Resources

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 Promega iPhone® app and use the Restriction Enzyme Tools to plan your next digest.

Double Digests? There’s A (Free) App for That

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®).

  1. Open the Promega App. Scroll down to and select “Restriction Enzyme Tools” or touch the “Enzymes” icon on the tab bar at the bottom of your screen. Continue reading

How to Use the Flexi® Vectors (Part 2 of 2)

pFN24K HaloTag® CMVd3 Flexi® VectorIn previous entries, I discussed the naming convention for the many Flexi® Vectors available from Promega before addressing how to choose which vector is appropriate for your use. However, I did not cover all the Flexi® Vectors available. In fact, I saved the HaloTag® Flexi® Vectors for this final installment. Continue reading

Efficient Cloning and Expression of High Protein Yields Using KRX Cells

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

How to Use the Flexi® Vectors (Part 1 of 2)

pFC7K (HQ) Flexi® VectorNow that we understand what the numbers, letters and abbreviations mean in the Flexi® Vector naming convention, how does one choose from among the over 40 different vectors? First I will detail the uses for the 21 vectors in the 1–12 range (plus outlier pF25) and address the remaining vectors in a second entry. Continue reading

Understanding the Flexi® Vector Terminology

pFN6A (HQ) Flexi® VectorWe work hard for our customers. Our various research groups are trying to find better, quicker and easier ways to purify DNA manually or using automation, to assay cell viability or apoptosis and yes, even to clone and express your protein of interest. Our Flexi® Cloning System is a simple and powerful method of directional cloning with a wide array of vectors suitable for many downstream uses, including adding protein expression tags, studying mammalian protein interactions, performing in vitro expression and expressing fusion proteins. Continue reading

Selecting the Right Colony: The Answer is There in Blue and White

cloning2Ah, 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