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?
1 day ago