Thursday, November 20, 2008

I must have fucked up syndrome

Its my own personal version of the impostor syndrome that others have talked about. I don't really believe that others will find out I don't belong there, its more that I will have to admit to myself that I don't belong here. That everyone was right when they said I should think about business or med school or anything but research. Despite all my protestations of enjoying it and being good at it, I really am not good at research. Which is horrible feeling because every thing I do becomes a confidence vote, proving to myself that I made the right decision. Case in point -Did my SDS gel not set - it must be something I did. The acrylamide went bad? Whew!!! Thank God it wasn't something I did, maybe I can do this.
This week it was the PBS that was fubarred. I had a bunch of wing disc dissections to do yesterday but the larvae were not co-operating. Everything had a weird consistency, the imaginal discs look "off" and I was just not able to dissect out the imaginable discs - they kept disintegrating. First I thought it was b/c I was hungry and hadn't done dissections in about 2 weeks, so just needed to practice on a few. After butchering 10 maggots, I had lunch and hoped I would get my mojo back. Nope something was up. Troubleshooting, perhaps I had put too much triton X in the PBS? After triple checking my calculations (seriously how hard is it to figure out how much 10% triton X to put into 100mL PBS to have a 0.1% triton x solution?), I eliminated that as issue. Maybe it was just a mojo / stars not aligned issue - lets retry in the morning.

This morning, I retried again, 3 maggots later I knew something was up. The only thing that changed between yesterday and 2 weeks ago was that I made up 10x PBS. Granted this was the first time in 3 years I made 10x PBS, but its not that hard to make. Especially Drosophila PBS, its 3 freakin ingrediants - NaHPO4, Na2PO4 & NaCl. Not hard. Only reasonable conclusion (in my head) I fucked up measuring out 3 reagents. I really really should not be here. OMG I'm Dr. Jekyls BF!!!
To see if something was wrong with PBS, I re-checked the pH of 1X PBS solution. PBS for flies can be anywhere from 7.0-7.6 and this solution ended up being over 8.0. WTF?? I had pH'd the 10x to 7.5. Granted I did not recheck the pH when I diluted it to 1X, but I have not experienced ddH20 changing the pH that much before. I must not have used the pH meter correctly (again because it somehow so technically challenging). How could I screw that up? Conclusion - Either the 10x PBS was pH'd incorrectly or I fucked up measuring the reagents so its not buffering properly.
Nothing was left to do, other than to remake the 3 ingredient 10X PBS, diligently checking and rechecking that I have the right reagents, the right weight, everything is completely dissolved before pHing etc. I added enough HCL to make the 10x PBS have a pH of 7.4. Guess what the ph was after diluting to 1x? Over 8! I diluted using ddH20 had been stored in a carboy, which is apparently very alkaline.

Long story short - the carboy h20 is very alkaline for some reason. Always check the ph when diluting down 10x solutions. The carboy H20 was what fucked up my PBS, not me. Whew! I am not blog fodder, maybe I can do this whole PhD thing!

Science never works, its all about the process and troubleshooting. I can not use the results of experiments, or the fact that something does not work as some sort of assessment of my capabilities. I really really need to learn that. You may read me saying that alot. I've read that if you repeat something 5 times before bed it will come true, whadday think?


Amanda said...

In that case, I'll have to try the 5x before bed thing, too. You're not alone, though. I always think that when an experiment doesn't work that I'm to blame. It's something I did (or didn't) do. Advisor once told me that I take too much personal responsibility for science not working (as in there are tons of variables) and chances are it's something that has nothing to do with me (like a former grad student mislabeling reagents). Maybe we should both take less responsibility for these things.

Mimi said...

I am really glad that you really are too hard on yourself. I'm with Amanda on this. There are too many variables so you shouldn't automatically blame yourself!

EthidiumBromide said...

I was always the same way too, until one day very recently I just woke up and stopped... caring. Gee, purification doesn't work no matter what I do? Eh, who cares? Not my problem. Nothing *I* can do about it. I'll just repeat it 50 more times and prove that it just wasn't meant to be purified in this way.

Obviously, this is just a temporary slump because soon enough I'll realize that it is VERY MUCH my problem or else I'll never graduate... but I have to say, for the time being, I am quite enjoying not beating myself up over every little thing!

The bean-mom said...

The best advice my grad advisor ever gave me was this: Don't let your self-esteem get caught up with any one experiment.

Very very wise advice. But it can be hard to take.

Good luck with your experiments next week...

Becca said...

Bless you for posting this.
I always feel so awful when I do something outstandingly dumb. (I beat myself up for weeks over running a gel with backwards polarity)
It does not make me less likely to be outstandingly dumb. Really, the only thing that prevents outstanding dumbness is getting enough food/sleep and practicing stuff regularly. When I do that I don't screw up PBS or the like. When I don't... yeah.

At least in the labs I've been in, we always depend on a *lot* of things working to get even the simplest data.

ScientistMother said...

Bean-mom - I think being emotionally detached from your experiments is very very wise advice indeed. I am learning that, but its a slow process.

Becca - Welcome to the blog! The funny thing is, if you read through the lab books of "brilliant" scientists, they are full of "silly" mistakes. I'm trying to keep reminding me of that. Its also a learning process