Who'd have thought?!

So, since I'm leaving the lab before my project is finished, I am hoping to hand it off to someone else in the lab. I had a meeting yesterday with one of the other PhD students to give hir a rundown on the whole thing. To prepare for the meeting I actually went back to read portions of my PhD thesis where I had written a discussion about what I thought might be going on and how to proceed and test those ideas. Now, I think 99% of people probably never open their thesis again after the exam, unless it's for a method or something. Usually if I hear someone re-read their thesis they find a load of errors/typos/things that just don't make sense, so I was expecting that. So I was pleasantly surprised to find I still liked what I'd written. It made sense, was well thought out and made some great points that I admit I'd kind of forgotten about over the last year or so. Who'd have thought? I worked really hard on my thesis when I was at a very difficult low point in my personal life. It was a major accomplishment for me to finish it and hand it in. I even had it bound in beautiful linen fabric cover in my favorite colour of sky blue. I'm so pleased to still like it.

On the experimental front I managed to confirm *most* of my antibody/construct results yesterday, just a few things to tweak a bit before I can move on to new things. I'm leaving for 10 days starting tonight so I'm looking forward to the break and coming back refreshed and ready to start fresh with new experiments.

Data outliers....

We had an interesting discussion this morning in a group meeting. Say you do an experiment on 5 independent days. Each day's data consists of multiple parallel replicates for each experiment, including an internal positive control. If one of the days shows that the internal control was actually negative, is it ok to throw away the whole data set for that day (because something was wrong with the assay?)? What if the other days showed a lot (or very little) variation? Does it make any difference if the experiment is a Western Blot, versus an immunostaining, versus a quantitative measurement (like cell proliferation values, or a luciferase reporter)? To be honest, I think I'd throw out that whole day as an off day and repeat it or use the n of 4 days... What say you?

In other news, I got wicked nice looking data with my new antibodies yesterday... today I'll try to reconfirm with antibodies against the tags instead of the protein of interest. AND I think there might be someone to take over my project when I leave, and s/he'd be the PERFECT choice - I'd be so happy to have this settled and start some work together before I have to leave. So, yeah!

Nothing works :(

Well, like the title says, nothing works! I feel like I've been busting my butt trying to get experiments to work. I'm trying to characterize a totally uncharacterized protein picked up in a loss of function screen with a super cool (if not so penetrant) phenotype. I made an antibody. And I'm trying (desperately) to figure out if it works, and where my protein is expressed and localized. Nothing works. Or it half works, or it just doesn't make sense and things are conflicting. Or the controls don't even work in the experiment. *sigh* So much for my group meeting/progress report this week. Summary: no progress made, oh except for the realization that I screwed up the cloning of my overexpression contructs and had to remake everything (at least I think I'm mostly caught back up with completed constructs). Not the best way to end my nearly 5 year stay in the lab (it's likely to be my last presentation before I leave in September). I am bummed. Why won't SOMETHING, ANYTHING work?

Struggling

OK, it's been a while again. I'm having a bit of an identity crisis. I always thought of myself as part of the "women in science" set, but these days, with my career on hold for my family, my project rapidly headed down the crapper, and my motivation dwindling to a near all-time low, I'm even a bit more saddened that 9 times of out 10 when I interact with someone else from the lab they ask me about my pregnancy and not about my work :( Is it because they think it's not worth talking about any more? I'm not one of them anymore? Or just an innocent curiosity about pregnancy?

I'm trying hard to make an effort NOT to bring it up myself because I don't want to be one of those women who can only talk about babies when they're pregnant. Of course it is an important part of my life now, but it's not the only part (or is it?) Most days I'd just rather go back to being "one of the guys".

I feel rather out of the loop though, in between PhD and postdoc, without any meetings/conferences coming up (except a talk I'm giving Friday at a local symposium), no manuscript I'm working on, a project I won't be able to see to its finish... I'm reading lots of other blogs out there with people counting down to defense, waiting for reviewer comments, writing grant applications and I just don't fit. I feel like I'm already "out" of science. And I'm sad about that. And I don't see when or how it's going to get better. I think back to the very kind email I got from one PI whose lab I applied to for a postdoc and she wrote that science will always be there for me when/if I want to come back, but I haven't even left yet and I still feel like it's already gone.

A little light reading....

I ordered new books last week and they finally arrived in the mail for me at work today! I'm so darn excited.



At least I have something to browse during all my washes and incubations this afternoon :p I think I have 4 of the 7 constructs and got halfway toward the other 3... I'll test digest and sequence today and I should have them finished up in time to transfect next week.

There is also some progess in getting approved to do some lab work (not that it's really stopped me from doing much so far...). A construction date for installing extra ventillation for the pregnancy lab is set for Tuesday next week, funding from the institute for buying some more equipment for the lab was approved last week and several things ordered, and a meeting with government officials is also set to happen next week. Yeah for progress!

Damn....

I realized yesterday while staring at yet more totally un-interpretable results that something must be not quite right. I went back through my notes and eventually, after some detective work I realized that I made a mistake. Months ago. A fundamental mistake. I mis-ordered two primers, one contains a stop codon, one does not (for making C-terminal fusions to my favorite protein). I copied and pasted the wrong sequence to the wrong primer name... and so I made all my constructs backwards. All the N terminal constructs aren't in frame with the tag and the C terminal ones are not fusions at all - they're untagged since the stop is intact in my protein. Crap.

This is big. I mean, it's easy to fix. I've already re-run the PCRs and will have the cloning completely re-done by the end of next week, but it means that all the experiments I did (and all the transgenics I made) since, oh, say, January or so have been completely and totally useless. I am so pissed at myself. How could I have made such a stupid mistake? And not noticed it? Yes, I checked all the sequences before using the constructs. They aligned perfectly with the vector files I had made, because I also made the vector files with the mixed up primer sequences. (D'Oh!)

I'm so embarrassed. And now I feel like trying to cover my tracks and hope no one finds out... that could be difficult seeing as I now have only 15 weeks left before the kick me completely out of work and I have to re-do about 20 weeks worth of work... of course at least this time around maybe things will actually make sense and won't have to be repeated 10 times before I can get some sort of interpretable result. This was not the brightest shiniest day in the history of my scientific career.... on the bright side at least it's me that found the error and not some poor project successor that got handed a bunch of reagents that don't work.

There go my plans for a relaxing weekend break from the lab....

decisions....

So, to update from my post yesterday, I was going crazy with all the what ifs, and so I knew I just had to take action... I'd been thinking for weeks and weeks, and it was getting worse, not better. So I sat at my keyboard and I typed out emails to each of the PIs that offered me an interview. I told them I was sorry for causing any inconveniences, that I had postponed my interviews earlier because my doctor had asked me not to travel until a safer point in pregnancy, and that now that it was becoming clear this little person was pretty darn likely to show up in November I was really not in a position to make decisions about big moves and new jobs. So I told them I decided to stay put for now, cancel the current interviews and re-apply for postdoc positions next spring. I heard back from 2 of them so far, both invited me to let them know if I wanted to re-apply whenever I was ready.

I'm still alternating between feeling like a huge disappointment to myself, my PI, and my colleagues, and feeling relieved that now I can just shift my focus to the work I have at hand in the lab, and staying healthy and reducing my stress level. Husband found a promising job ad to apply to and has spent the last few days re-vamping his CV and putting together all the other paperwork they're asking for. Let's see how that turns out.

I wrote a long time ago that I was reading the books Mama, PhD and Motherhood: the Elephant in the Lab and that I would post reviews when I was done. I finished the books ages ago, but haven't been able to gather my thoughts enough to write a review. Maybe I'll still get to that. But for now, let's just say, when I first read the books, I was so disappointed. So disappointed for the women who couldn't have it all, who made major sacrifices, either in their career or in the way they raised their families to try to just do the best they could... and now I feel like I know a bit how hard those decisions must have been and how complex. And I have new-found respect for those brave enough to contribute their stories.