Fast and Affordable Ligation
by Sven Löbrich, Labtimes 07/2013
Speed up your ligations with a home-made buffer and invest the saved time and money in an afternoon at the beach or in the next bar drinking margaritas.
Ligation buffers are usually mixed from five simple ingredients. Only one, however, has a major impact on the speed of the ligation reaction: Polyethylene glycol.
If you do a lot of cloning in your lab you may at times wonder how to make your ligation reaction faster, more reliable and at the same time much more affordable. Even if you haven’t, keep on reading – it’s never a bad idea to save time and money. I, for one, can think of a million things to do if I had copious amounts of both these (unfortunately rather rare) commodities. Going to the beach and drinking margaritas is usually at the top of the list. So, how can you save both time and money to make that happen? The answer is so simple, it hurts to look at it: go fast and go cheap!
There are a lot of expensive ligation kits out there. The so-called “rapid ligation kits” are popular because of the added value that the ligation reaction needs neither a controlled temperature, nor a prolonged incubation time. Conventional ligation requires incubation over night at 16 °C. With a rapid ligation kit you can get away with as little as ten minutes at room temperature – dang!
True, these will save you time – but they won’t save you any money. Which means you can go to the beach but you won’t be able to afford the margaritas! So, you might as well stay in the lab and continue with your experiments.
The reason why the rapid kits work is not because of some fancy Speedy-Gonzales-ligase that is capable of doing what the regular ligase can’t do. Turns out it is exactly the same enzyme, i.e., good old T4 DNA ligase. Rather, the secret is in the buffer: because it is very viscous it slows down the diffusion of the reaction partners, effectively mimicking the effects of reduced temperature. This increases the chances of loose DNA ends matching up, sticking together via their overhanging unpaired nucleotides and getting ligated. Because the sample is not chilled the actual enzymatic reaction is not slowed down; which is why ten minutes are indeed plenty of time for the ligation to occur.
I have applied this protocol literally on hundreds of cloning experiments and it didn’t fail me once. All we need to know, you might say, is the composition of the buffer. Manufacturers of rapid ligation kits will rather cut off their right arm than tell you what’s in it. Luckily, my sense of self-destruction has not quite developed this far – which is almost surprising given that I have spent the last fifteen years in research.
The key ingredient that will make all the magic happen is polyethylene glycol (PEG). PEG is an inexpensive polymer that is sold as a dry powder. Depending on the average molecular mass, you can get different PEGs, ranging from 3,350 g/mol to well above 20,000 g/mol. Usually, the lower mass PEGs do well in this buffer.
Here is the recipe:
- 132 mM Tris, pH 7.6 with HCl
- 20 mM MgCl2
- 2 mM DTT
- 2 mM ATP
- 15% PEG (3350, 6000, or 8000)
This yields a two-fold concentrated buffer. Since the buffer contains ATP and DTT it’s a good idea to aliquot and store it at -20 °C and avoid freeze-thaw cycles. For an average ligation reaction I usually aim for a total volume of 20 μl, ligating 100 ng of purified vector backbone to three times as many insert molecules. Hence, a typical reaction would look like this:
- 100 ng purified vector
- 3x molar ratio of purified insert
- Add ddH2O to 9 μl
- 10 μl of 2x homemade ligation buffer
- 0.8 μl of T4 DNA ligase
Vortex briefly, collect by centrifugation and incubate at room temperature for ten minutes. That’s it! Now you can chill the reaction on ice, add your competent cells and proceed with the transformation. This also means that you have just saved a lot of time and money.
Therefore, pack your beach towel, grab your swimming trunks and off you go to “Margaritaville”. Enjoy!
For more detailed instructions, check out this link: www.benchfly.com/video/61/make-your-own-rapid-ligation-kit/.
Last Changed: 25.11.2013