binary|fusion » HowTo

cloning trick: ligation of multiple inserts

adamcoster — Tue, 12 Jul 2011 02:44:23 +0000

I’ve spent the last couple months building a plasmid library, and in the process I thought of a trick. Ligations, perhaps the worst part of cloning, are notoriously finicky reactions. The goal is to take several pieces of linear DNA, where the ends of the pieces can only connect in a certain way, and then use an enzyme (T4 Ligase) to sew them all together into one piece (in my case, a circular plasmid).

Figure 1. Ligase (2HVQ.pdb) rendered in PyMOL. Click to see a crappy animated GIF!

I needed to insert three fragments at once into a single backbone. In my ignorance (from my lack of experience) I thought ligating four fragments should work just as well as two, so I just threw them all together and ran the reaction. The result was a mess, and when I tested 40 different clones afterwards not a single one was correct. So I started adding them one piece at a time which, obviously, was going to take three times as long.

The next morning while in the shower (one of the best places for random ideas) I thought that, though most of what was in my test tube was not the desired product, there was most likely some tiny population that actually was the correct fragment. The problem was just that putting my products into E.coli and then trying to find the correct thing wouldn’t work if only 1/100 (or less!) of the ligation products were correct. But PCR is wonderful for making tiny amounts of DNA into large amounts, so I thought, why not just try to use PCR to get the correct thing out of the mess? Since each product is made out of some combination of the input fragments, the lengths would be discrete and therefore visible and, potentially, separable on a gel. I just needed to be able to copy up enough that I could see DNA run on a gel, and then I could just purify out the one that was the right length.

Figure 2. Each differently-colored fragment is being ligated into the gray backbone. The primers (black arrows) flank the entire insert.

I designed primers that would flank the combined insert and did a PCR (I can’t say “PCR Reaction” for the same reason I can’t say “ATM Machine”), and did indeed get a band of the desired length! After gel purification I was able to ligate the entire insert into the backbone without any trouble.

You might be asking, “Well isn’t this now a two-step process, just one less than the three required otherwise, and so barely worth the effort?” Almost, except that there is no need to transform the multi-fragment ligation mixture. So it’s more like 1.5 steps. Plus, the ligation, PCR, gel purification, final ligation, and final transformation can all be done in a single day!

Though my labmates were surprised when this worked, it turns out I was a year too late to publish this idea . If you are fortunate enough to have access to Analytical Biochemistry (another journal that should be free to the public but isn’t), see the paper by An, Wu, and Lv called “A PCR-after-ligation method for cloning of multiple DNA inserts“. If you aren’t that lucky, I’ve already told you everything you need to know!

Mindforge Tutorial: Cleartype Tuner

adamcoster — Sat, 30 Apr 2011 12:00:17 +0000

As part of the continuing closet-cleaning series: WindowsXP text is ugly and induces eye-pain and headaches. Here’s what you can do about it:

Mindforge Tutorial: Try out Linux using Wubi

adamcoster — Sat, 23 Apr 2011 15:40:30 +0000

Still cleaning out the closet… I made this video tutorial a couple years ago for my brother‘s and my short-lived computer company. It’s a little outdated, but (probably) still accurate.

Mindforge Tutorial: Disk Images

adamcoster — Sat, 16 Apr 2011 12:00:20 +0000

If your first thought, when reading the title of this post, was “What in the hell is a disk image?”, you probably aren’t alone. But before you decide that you don’t care enough to read on, let me quickly tell you what the big deal is so that your decision to stop reading will be an informed one!

A disk image is a virtual copy of a real disk (so a virtual CD).

That’s it. This is cool because you can make backup copies of your CDs/DVDs (though some have protections that may make this difficult), video games, operating systems, etc etc.

On top of that, you can use virtual CD-drives to play your virtual disks! This has a few advantages: [1] your computer communicates faster with your virtual disks than your real ones, and [2] you won’t have to worry about carrying CDs around ever. Of course, CDs are going the way of the Dodo, so this post may already be useless.

In any event, my middle brother and I once started a custom-PC company called Mindforge Technologies. I made a screencast tutorial for the company that shows how to use disk images, which is the Youtube video above. Enjoy!

useful bioinformatics resources

adamcoster — Fri, 21 Jan 2011 18:00:35 +0000

I’ll try to keep this page updated with the kinds of things that I’m familiar with and find the most useful. The resources in this list will be used extensively in tutorials on this site.

binary|fusion » HowTo

cloning trick: ligation of multiple inserts

Mindforge Tutorial: Cleartype Tuner

Mindforge Tutorial: Try out Linux using Wubi

Mindforge Tutorial: Disk Images

useful bioinformatics resources

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