InaDWriMo 2008 progress report and other fun stuff

Well, according to my status bar, I am 40% of the way to my projected goal for InaDWriMo. However, progress is temporarily halted since my boss wants me to stop writing. I told her OK, but I am writing in my free time so I think I'll stop at the end of the month.

Now for the other (not so) fun stuff.
I am actually in a shitty mood because things aren't working. There is a month remaining in gradstudent lab and I need to finish more than a months worth of stuff. When I defended my dissertation in September, I thought that there was ample time, but that was before the triple mutant from hell.
I need to make a triple mutant lacking genes A, B and C. Right now, I have double mutant AB and single mutant C. I've tried introducing the knock-out construct for deleting genes AB into mutant C, but it is toxic and the cells get really pissed and lose the KO construct before homologous recombination can take place. I tried to get around this by growing the cells in the presence of antibiotic since the vector contains resistance to one antibiotic and the genes are being replaced with a cassette that imparts resistance to another antibiotic. I obtained many colonies with the correct antibiotic resistance phenotype, but all of them still contain the genes I am trying to delete. The number of colonies and the rate of growth of said colonies indicate that they are not spontaneous, antibiotic-resistant mutants, indicating that the resistance gene is somewhere in the chromosome. Where? I don't know. I only know where it is NOT.

Obviously that wasn't working out, so I tried the opposite approach. Delete C in the AB double mutant background. Fine. I introduce the KO construct and the cell grow normally (yay) Unfortunately, that is the end to the good news. The KO vector for C is a temperature-sensitive vector and gene C is only deleted, not replaced with an antibiotic-resistance gene or cassette. So, it is screening hell. This approach works in the following way: The vector is integrated after growth at a non-permissive temperature. Cells are returned to the permissive temperature and and are passaged several times. At some point dilutions are plated on media containing no antibiotic. These colonies are replica plated onto additional plates that do contain antibiotic. Those that are sensitive, are screened for the mutation using PCR...colony PCR.
Unfortunately, the locus I am deleting doesn't seem to amplify using colony PCR, so I ended up trouble-shooting PCR. PC fucking R people. This is like trouble-shooting shampooing my hair. Finally, I found primers that work, except they don't yield a product if the gene is deleted. Awesome. A fucking negative result. This means that I have to recheck any colonies that don't yield a product and so far, all of those potential candidates have yielded a product the second time through. Still, it is better than growing up 5 zillion cultures and extracting DNA for PCR. Now, I am adding an antibiotic-resistance gene to this construct, so that I will have a selection. Good thing I am a rock star at cloning.
I am also considering that this triple mutant is lethal, although it really shouldn't be. We'll see. I am going to give it the ol' college try one last time with the new vector and see how it goes. If I can't get this mutant, then I will just have to drop the strain from the manuscript. :(

So, yeah. I am irritated to say the least.

Oops. I forgot about the InaDWriMo Update.

I somehow missed that I was supposed to update my InaDWriMo progress on Saturdays. It doesn't really matter though, even if I had known, I wouldn't have posted an update since I was busy taking engagement pictures and partying.

My oldest friend, who happens to be a professional photographer, came into town to take the engagement pictures. In all honesty, I wasn't 100% on board with taking these pictures. Mainly this is because I think most engagement pictures are completely stupid. The poses are so ridiculous and awkward, I feel embarrassed just looking at them. Two people gazing into each others eyes makes me want to puke. On the other hand, Lancelot and I don't have any nice photos together, let alone any professional ones, so I'd hate to miss an opportunity to get some.
After complaining to my friend, she said she would be happy to take the pictures for me, and this helped tremendously. First of all, since she is a fine arts photographer instead of a wedding photographer, the pictures would not be posed and awkward. Second, I know her well, so I am not going to feel like such an idiot during the session.
Everything ended well. Thanks to having my friend take the pictures (and 1/2 a xanax), I didn't feel completely silly. I can't say I love the pictures, but that is mostly just because I am in them and really, nothing can be done about that. (One of the extra pictures is shown.)
Immediately after the photos, we all went to a party where I drank a little too much. So, as you can see, there was simply no time for an InaDWriMo update.
Now for the update: I haven't accomplished much of anything in the way of writing. I really want to get a serious start this week, but who knows if that will happen. I expect an insane surge in word count toward the end of the month, since I seem to enjoy waiting until the last minute to get anything done.

Write my ass off...at least that is the goal.

At the end of 2008 I am saying so long to graduate school lab, but before I go, I need to bust out two manuscripts. I procrastinate like no other, so I need some extra special motivation. I am hoping that participating in InaDWriMo will provide said motivation.

Wtf is InaDWriMo you ask? It is International acaDemic Writing Month. It is hosted over at Dr. Brazen Hussy's blog. If you want to sign up or get more info. you should check out her post.

The month-long science write-a-thon started on Nov. 1. Yeah, Nov. 1 was a Saturday and starting a month-long venture on a Saturday just seems inherently evil. Really, starting a long project on the weekend is probably a bad idea, so Sunday was out too. I am not sure what happened on Monday, but it had something to do with the status bar and finding it. My Tuesday evening boot camp class was canceled, so I decided to officially start participating tonight while half watching the election coverage.
I placed the handy little progress bar on the right hand side of the blog. Feel free to kick me in the ass if it doesn't move. Basically, I am hoping that by publicly stating my goals, the embarrassment of not reaching them will drive me to get some writing done.

My goals are simple. Write two manuscripts, including abstract, introduction, materials and methods, results, discussion and figure legends. I want to accomplish as much as possible so that when I move to post-doc lab I can focus on learning scary biochemistry.