Make Your Own Rapid Ligation Kit
by
I did a lot of cloning as a graduate student, and even more as a postdoc. In total I’ve now cloned somewhere around 300 constructs. As we all know, one of the real bottlenecks in cloning can be the ligation reactions. Using traditional ligase, it takes three nights (set up ligation, transform bacteria, pick colonies) to get to purified DNA. And this is assuming we haven’t had to insert any black magic ceremonies to get the reaction to work in the first place.
The development of the rapid ligase kit was a major step forward because it reduces the ligation reaction to 5 minutes, cutting an entire day off of the process. (So, 300 clones x at least that many reactions x 1 day saved/reaction = a job before I’m 90?!!?)
The problem was that soon I realized I was either going to need to revert to the traditional ligation reaction, or bankrupt the lab with all of the rapid ligase kits I was going to need to purchase. There aren’t enough promotional codes or vendor discounts out there to make cloning 300 constructs with a rapid ligase kit affordable. But I had heard that the secret to the rapid ligase kit was actually in the buffer. So I started creating my own cocktails, trying to see if any of them would work. This video contains a recipe that works like a charm. 300 clones later and I’m still going strong…
One evening we were filming “How do you say ‘Hoechst’” and (not surprisingly) I was setting up a ligation reaction in between takes. We left the camera rolling and shot the rapid ligation kit video in one take. Although there is no tricky German pronunciation in it, I hope you will find it useful all the same!
