Daily challenges in the lab

Ah, such is the life of a scientist, where nothing, nothing is straightforward!  Science never sleeps; it is like a never-ending unfolding story, with as many twists and turns as you like and then some.  Recently, yours truly has been going through a rough patch in the lab of late: a ligation experiment that would allow for some wonderful and intriguing discoveries isn't working, the mutant yeast strains I have generated are not what they seem.  I am repeating some stages in my experiment that I did three months ago.  In the meantime, other experiments I really want to do to get things moving are on hold while I try to find out what I am actually working with.

What the hell are you?! Answer me, damn mutant!!

 If molecular biology was a video game and there were bosses - gigantic monsterous enemies you must battle to get to the next stage (not your supervisor, although opinions may differ on this point) - then ligation would definetly be one big bad boss!  The idea of cutting up pieces of DNA with restriction enzymes and sticking them together with DNA ligase sounds simple enough, but there is a lot of fine tuning involved.  The ratio of vector DNA versus insert DNA is important, get this wrong and it might not work.  The ATP in the ligase buffer needs to be in good condition as this is needed by the enzyme to function.  The temperature needs to be right, the salt and pH conditions need to be right, your mood needs to be right, the funny dance that you do with the pirouette at the end to ward off bad luck...right! 

Giant, evil-looking ligation boss: 1 million points ; tiny, suffering scientist: none

But for now, something is amiss and I need to find out why so I can move on.  One of the frustrating things about science is time: it takes a good few hours to digest the DNA, half an hour to clean it up, half an hour to prepare it for ligation and then another hour for the actual reaction.  It can be so frustrating to find out after all that time it hasn't worked and you have used up loads of enzyme, tons of DNA and it is late in the day so there is little you can do about it - apart from huff and puff all the way home.

So, good people of the blogosphere, if there are any scientists out there who can impart a little advice on how to make ligations work, please feel free to sprinkle me some crumbs of wisdom.  Otherwise just wish me luck - I sure could do with some!