March 1st, 2007

Raupe

shRNA

Has anyone of you used the vector pLVCTH tTRKRAB in vivo? I want to use it for my Master thesis project but I've heard from two other labs that they didn't get good knockdowns in vivo and most of the time their shRNA got silenced and they weren't able to induce it at all. The vector has been published and the experiments in the paper suggest that it should work fine. I'm a bit insecure now if I should use this vector or rather look for some other way to do what I want to do.

Thanks for any comments.
PSU

Time to rant...

Oh contamination, how you have been plaguing me (pun intended, considering we work with Y. pestis).  So I figured that the plates that were contaminated a week or 2 ago was due to using contaminated saline.  (I'll blame the co-worker for contaminating it).  But why our plates were contaminated today, I have no clue.
Ok, so here's what we did:  Injected rats with a dose of a certain Y. pestis strain, that was supposed to be around 10^7, but I don't know because my dilution plates were too overgrown, and I didn't dilute down far enough.  At 48 hours, the rats were euthanized & necropsied (is that a word?).  We removed certain organs/pieces of organs, placed them in 5mL of saline (neat), and then ground the organs with the "Tissue Tearor".  Then, we took 10uL of the organ slushie, and put that into 1mL of saline (dilution 1).  Mix well, take 10uL of that dilution, and put into 10mL (dilution 2).  Mix well, ect, etc.
Then we plated 500uL of the organ mixture onto yersinia select plates, and 100uL of each dilution onto plates as well. But for the 2nd dilution, we used BHI plates for some of them.  Probably not the best idea, because most of the BHI plates had a load of contamination, like completely covering the plate, but some had individual colonies that could be counted.  But nothing grew on the dilution 1 plates, and some of the neat plates were contaminated.  So, I'm guessing the saline was not contaminated, but if the original sample was contaminated, then the 1st dilution should have some contamination, right??  
So where the hell is the contamination coming from?  I'm not a very experienced microbiology person (I'm still learning how to make up a solution to get a particular O.D., and if my boss says to use a solution that's 10^4, how to figure out what O.D. that should be, etc. ).
Any advice/comments/criticism???

I'll just blame my co-worker, who is probably one of the worst people I've ever worked with.  No, he's definitely the worst.  No concept of sterile technique, likes to just do his own thing, doen't listen, pays no attention to detail whatsoever, is f'ing lazy, and lies like no other.  We had a rat die because its water bottle was empty...he claimed to have checked them all the day before, and wouldn't admit to not checking it.  I'd rather you confess to not doing something than flat out lying to my face.  Oh, and rats do not drink 500mL of water in 3 days, despite what  you may think.  I had to ask him 3 times to make sure to order nitrile gloves, size small.  He CC's me on the order email, asking for small latex gloves.  For awhile, I had to email him every week to tell him what needed to be done.  For someone who's been working a long time, he should know (I'm older than him by about 3 years, but he didn't go to college.  This is also the kid who was making about as much money as me when he started working.  Me, with the college degree.  And him, with just a high school degree and piss poor work habits.) 

I think the poor kid gets yelled at daily, and threatened to quit a few times, because he could make more money somewhere else...but he probably wouldnt get the flexibility he has here, leaving early to go to class, etc.  The worst of it was him lying about his timecard.  See, he works for us (the Army contractors) full-time. Since we share our suite of rooms with the Navy group, they offered to pay him to take care of their animals, getting paid hourly.  Now I don't know about most people, but it should't take 15 hours/week to change cages.
I happened to see his timecard on his desk (No, I wasn't snooping!!), and saw that he filled in hours for days that he was supposed to be on vacation.  He did this again on his next timesheet...getting paid for hours he did not work.  F'ing liar!!  So, I had to call him on it.  What did he do?  Get mad at me for looking at something on his desk.  Hey, I can't help it..I have to walk past it to go to the microwave!  
But his boss from the Navy had a little talk with him, but I have no idea how that went.  Hopefully he gets his shit together but I'm not optimistic.  I just don't like the kid, don't trust his work, and don't believe 90% of what he tells me.  (He once asked me to read a paper for his class, and as I was reading, thought to myself that there's NO way he wrote this himself.  The grammar & sentence structure was just too good.  I've read his poorly worded emails, and I know his English isn't that good.  So did he plagiarize or have someone write it for him?  I know that spell & grammar check in word isn't good enough to fix his mistakes..
And I'm basically in charge of him.  Woohoo.  But at least I got a big raise out of it!
DISGRUNTLED

(no subject)

Dear undergrads please refrain from burning the lab down, This can be done by leaving boxes of kimwipes on the hot plate, or if you do atleast make sure it is turned off. 

Yes this happened, luckily it wasnt hot enough to start a fire in that short of a period in time. The hot plate (I am not sure if that is what it is called) is kept warm, but not scalding and on at all times because it takes forever to heat up. I am not completely sure it would have started a fire, but the thought of the lab burning down this far into my work freaks me out.