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Cloning Round Up

Posted in: DNA / RNA Manipulation and Analysis
Cloning Round Up

Cloning can be the bane of a scientist’s existence. Rarely is cloning the end goal of an experiment. Generally it is the means to an end for studying something interesting that piece of DNA does, such as expressing a protein. That’s where we come in. We have tips and tricks for all sorts of cloning. This time I’m rounding up some of my favorite articles on tips for cloning using ligation.

First up: Before we get into tips for cloning success, let’s talk about how DNA ligation works. Demystifying the DNA ligation process makes troubleshooting, optimization, and all those other tips and tricks less confusing.

Now, I don’t know about you, but I’m always looking to save time in the lab. Streamlining your cloning can save you many hours! Making my own “pre-cast” agarose gels saved me many hours.

Next, what happens when things go wrong? We troubleshoot! These are controls that I still do when I perform a ligation reaction—and I insist that my undergraduate students do them as well.

There’s always room for improvement, though. That’s where optimization and better ways to cut your plasmid comes in. Tweaking some of these variables can greatly improve your cloning reaction.

Let’s say all this optimization doesn’t work. Sigh. That seems to be science at times, but never fear! We have some tips and tricks up our sleeves. Did you know that the DTT in ligation buffer is volatile? If your ligation buffer doesn’t smell like wet dog (as my grad school advisor always said), then it’s time to get a new aliquot.

In the end, we still need a way to screen for our product. It doesn’t have to be pretty, though. Sometimes all you need is a quick and dirty screen before sending your product off for sequencing. As a side note: always send your cloning product off for sequencing. In one infamous case in grad school, I had a duplication event that changed the length (and sequence!) of my protein.

All this cloning works up an appetite and you may need to step away from the bench to grab some lunch. That’s where it’s useful to know where you can pause your cloning reaction.

This is just a sampling, an amuse bouche if you will, of some of my favorite cloning tips and tricks. What are yours?

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