Fig.1: Kit contents.

Since I intend to do a lot of fiddling with Arduinos, and because I need the soldering practice, I bought the breadboard voltage regulator kit (by Solarbotics) from Makershed. The kit is simple – it just requires the soldering of a handful of parts to the provided board. The instructions are super-clear, so even though I barely know what the parts are and only recently discovered that LEDs have to be connected in a certain direction, the task was easy. I did find, however, that I still totally suck at soldering, even after the purchase of a nicer iron. With time perhaps.

In the end, though it took me probably 5 minutes to solder each joint, I plugged the thing in and it works! This regulator is supposed to hold the voltage at 5V, which is perfect for the Arduino since that is what it outputs. Pretty handy little thing, and if you are a bit more knowledgeable you could make your own from scratch for way cheaper.

Fig.2: Assembled kit, plugged in with glowing LED.

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!

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!