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 “Cloning Tips for Restriction Enzyme-Digested Vectors and Inserts”

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