Monday, June 11, 2007

second PCR and second primer

...and the second primer worked the first time I used it as well. With the correct temperature too. Wow! I am feeling content and happy now, yet I still believe something is wrong. Or at least it will not work all the way... ah well, I'll skip my [realistic] pessimistic view of it all and try and purify the pcr products tonight so I can run the third [VERY] important pcr tomorrow morning. Actually, I am pondering staying in lab for another four hours and do the pcr tonight but nahh... I should sleep some and then of course it's the dreaded 'being tired and sloppy' which I really don't want to be today! Not when it is working!! (small dance of joy!!)

off to clean the products.

update> I stayed (not really surprised) and put a reaction over night. I did, however, elute the purified products into 50 microliters .... I should have remembered that concentration is the key. Tomorrow I'll redo it all and remember to elute into 25 microl maybe? hm, I'll think about it. All of it probably since I didn't gel purify but rather did a quick pcr prep purification. I wonder if that is a big difference, really? Anyone knows?

Friday, June 08, 2007

Experiments at Friday afternoons

The pcr worked! I am so happy with myself since the pcr with the new (correct!!) primers did work the first time. With both the strains. With the higher temperature! Go me. Or something... The only thing that is annoying is that the second (new) primer, i.e. the second primer in the second pair of primer pairs that I need, have not been synthezied yet. The tech mumbled something about ”low yield” or something along those lines when I went down to the facility Wednesday to ask why the third new primer hadn’t been with the other two. Well, hopefully I can have them Monday and if (cross my fingers and wish for everything I hold dear at the moment) the reaction works on Monday I might actually greet my boss back with a happy ”I think I have the mutant”.

This is of course based on the fact that not only the first pcr reaction needs to work but the second one, with the three different fragments and the three different primers need to work when put together. Then the transformation (which I have yet to get to work in the other strains with that other fragment) must work. And then of course the cultivation and examination. But hey, I am going to feel happy for my wonderful band on the TAE gel today since it is Friday and doing an important experiment the last few hours of the day might result in a very distressed post doc for the weekend or, as in this case, a post doc that looks onto next weeks bench work with happy eyes and hope in her heart. This based on the fact that I am not considering the looong time spent trying to amplify the fragment using nonoptimised primers and other smaller things that have made this take way too long time. Not thinking about that now. Actually, it might not be that important since I learned what I did wrong and redid it right. Or so I hope. (knock wood!)

How mushy is this anyway? To be honest though, about the life of a post doc: I still have to go in to work later tonight and tomorrow morning and then tomorrow night and then the morning after... well, you get the picture?! Time points. As can be read about
here [shamelessly proud is what I am about it!]. Hopefully that experiment will turn for the better because at the moment those things are looking a little bleak. Ah well, at least I might might have half a mutant ;) Time to go home for running and then dinner before returning to work and donning the protective gear.

Tuesday, June 05, 2007

-80 degrees = burns

Having a PhD does not automatically make you smart. I remember I used to tell friends back home this after they pointed out that it was strange that I did this or that. Today I got reminded again. When opening the [vertical] -80 freezer to pull out the racks in metal it is better if you a)don’t press the side of your hand directly towards the metal in order to keep the rack stabile and b)don’t think ‘this will only take a second’ but then realising that the box you want is very close to the back of the freezer. IF one wouldn’t remember this (who could be that stupid?) one could end up with a burn on one’s thumb. But of course, this is just hypothetically speaking. No Doctor would ever forget to put on the BIG gloves when handling the -80stuff. Nahh…..