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®).
- 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.
- Select the first enzyme of your desired pair. You can search by name of enzyme or cut site. You can scroll, use the alphabetical list at the side or type in the sequence or enzyme name.
- In this instance, I chose EcoRI. I get a diagram of the palindromic cut site, the overhang produced, the activity temperature, the buffer that is supplied with the enzyme from Promega, and a list of the buffers in which the enzyme is active. If the enzyme has star activity in a buffer, the buffer is shaded with a magenta color. If I want to perform a double digest, all I need to do is touch the square at the bottom of the screen.
- After you touch the double digest box at the bottom of the screen, you will be able to select your second enzyme. You can control expand or contract the list of available enzymes based on the amount of desired activity you desire in the second enzyme.
- Choose your second enzyme and you will see a screen that compares the activity of the two enzymes in all of the available buffers. In this case, I am looking at a double digest using EcoRI and Xba I.
And, what a delight, I can perform the digest at 37°C in buffer H. Both enzymes will have 100% activity and no star activity. You can download the free Promega mobile app with restriction enzyme tools here.