Cloning trick: ligation of multiple inserts

[2013.02.26 Edit: A number of people are finding this through Google searches. I don’t have an updated post on the topic, but if you’re trying to assemble multiple DNA fragments then I suggest looking into Gibson Assembly. NEB sells* a dead-simple mastermix, which is a bit pricey per reaction (I just make my reactions half the size) but comes out to cheap when you take into account the cost of labor (so long as your PI values your time…).]

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.

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Notepad++ and Regular Expressions

A few years ago I wrote a post on making Notepad++ your default text editor, wherein I expressed my extreme love for this little piece of open source software. To date, I use it for just about everything and, after extensive use in a computational biology lab rotation, I figured I should share one of its most amazing features: Regular Expressions.

I’m not going to take the time to define Regular expressions (“REs” from now on) and will instead send you to Wikipedia. Just know that they are totally badass. In a nutshell, REs let you do really fancy search-and-replace in a text document. Perhaps that doesn’t excite you, but let me give some hypothetical situations in which you may find yourself:

  • You have a file of contact information for everyone you know (say, 1000 people) and want to get just the email addresses so that you can spam everyone.
  • You have a large FASTA file and want to pull out all of the organism names.
  • You want to convert a file from one format to another.
  • You want to combine multiple lines into a single line.
  • You want to separate a line into multiple lines.
  • Other pain-in-the-ass sounding stuff.

Sure, you could manually copy-paste all of those email addresses or organism names, and you could go through and hit the ENTER key to put things on separate lines. OR, you could write a few characters into NP++’s Find & Replace box. I think an example is the best way to make this work.

As a biologist, I’m rather fond of FASTA files. FASTA is simply a way to format DNA or protein sequence data so that people and programs can easily do stuff with that data. The format is:

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More Puzzling

In a previous post, I discussed my attempt to write a program to solve a puzzle. I never updated that post because, well, I ran the program all night and it didn’t find the solution!

I had made up a fake puzzle that I knew had a solution for testing, and the program could solve it in 15 minutes. But it couldn’t solve the one I had recorded for the real puzzle. I figured (and hoped) that I had simply recorded it wrong and to check, I re-recorded the pieces and tried again. And it worked! Here’s how:

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Homeopathy: Belly Flop to a Cure!

Homeopathy says essentially this: Something that causes disease can be made to cure that same ailment if the something is diluted into water so much that it is no longer present. For example, if you were to take one molecule of cyanide and dilute it into a swimming pool, then drink some pool water, you would be cured of cyanide poisoning! Sound too simple to be true? Well, that’s because you must first release the Energy of the cyanide. Personally, I do this via belly-flopping into the swimming pool, though any kind of concussion of the water will do. Punching the water is also a pretty good way to do it.

Another example: it is clear that humans are basically a disease of the earth. We’re polluting the crap out of it, killing all the wildlife, etc. Now, this should be easy to fix with homeopathy. Since people are causing these bad things on Earth, all we need to do is have someone go swim in the ocean, concuss the ocean, and then distribute ocean water all over the world. In the same way that you can dilute a cyanide molecule into a pool, you can dilute one person into the ocean. You still need to release the Energy of that person, though. I would suggest the concussion be from underground faults leading to earthquakes and tsunamis, though enough belly flops would likely also do the trick. I feel quite certain that there have been a few people in the various oceans when these kinds of concussive events have occurred, therefore it is clear that Sea Water can already fix global warming!

And while we’re thinking about it, just imagine how many things have been diluted into the oceans: people, animals, plants, drugs, chemicals, pesticides, fish nets, volcanoes, tsunamis, airplanes, boats, etc, etc, ETC! This means that sea water can stop volcanoes from erupting, release fish from nets, repair damaged boats and aircraft, fix broken bones caused by tsunamis and downed aircraft, release the hallucinogenic hold of LSD, cure various cancers, and so on. In fact, if you have a disease, all you have to do is jump into the ocean, and then you will be diluted to homeopathic levels, meaning your disease will be diluted to homeopathic levels.

Belly flop into the ocean. Then drink up. Any disease you have will be cured!