Ha-ha-ha-ha, Luria broth! Just do me a favor, when you write up your results, please be sure to introduce your acronyms so I don’t have to e-mail a query, for example when you use RT (reverse transcription? reverse transcriptase?). I am SO tired of having to guess what a microbiologist is saying in his friggin’ paper! (I edit microbiology journals for a living, so I actually understood the OP, go me!)
If you still have the tube try running about 5 ul of water in it and transfecting that… there is almost always 5-10 ng dried on the wall and that is enough to transform with. YMMV…
The insect cells grow great… the hard part is getting the little buggers to express the protein.
Good luck and at times like this I reflect on what a college prof told me after having a bad week trying to get a PCR reaction to work… “Molecular biology is alot like voodoo, you have good days and bad days” 
Since reading one of CRorex’ rants a while back, I am always reluctant to open his pit threads. There are all these deadly microbes floating around, surrounded by human incompetence (not CRorex’, but some other individual) - a potentially lethal combination.
CRorex is the only member who I think requires a special rating system to warn the reader of the scariness of the rant. I propose the following:
RED - some assmunch ALMOST destroyed human life today through his incompetence.
YELLOW - some assmunch ALMOST unleashed a virulent plague that would have wiped out half the human race through his incompetence.
GREEN - some assmunch screwed up CRorex’ day through his incompetence.
This would allow the reader to brace for the degree of potential disaster.
This could have easily been me last summer. (Imagine me banging my head against a desk after my thousandth gel wih no PCR results: “WHY… WON’T… YOU… AMPLIFY?”)
Now I just have to sort through eighty-odd RFLP gel pictures and figure out how they’re related and whatnot. Fun, fun…
- tsarina, another frustrated microbiologist.
Someone owes us for 2 uL of Sal 1 restriction buffer. But I think that’ll help.
Hehe, personally I LOVE cellular virology. Doing tissue culture work, primary tissue work, growing virus and all that other fun stuff. I can do a little bit of everything except for westerns. YAY! No western blots of me!
And I swear ALL science is like Voodoo. You’ll have 9 months of EVERYTHING working the first time. Then 2 months of everything but the gaslines exploding in the lab.
Particlewill: There’s absolutly nothing to worry about bacterium with viral plasmids in them. The plasmids are made so the bacteria won’t express the viral DNA.
Hardygrrl: Hehe 
Gobear: RT can be both of them 
Generally RT should be for reverse transcription and used as protein/kit names. Reverse Transcription and Reverse Transcriptase can more or less be used interchangably. “Using RT we generated the dDNA”. Personally I’ve never been able to keep in vitro and in vivo straight
Cards: Hehe!
I know your pain. I was trying to run RT-PCR for some cell surface marker that my prof believed was linked to Lupus.
So we had two species of mice we had total RNA (spleenocytes–sp?) from. I’d get the primers and run reaction after reaction.
Well, it turns out that 1) we had primers from the wrong SPECIES. 2) The mutation we were looking for occured in the beginning of one end of the gene (I think)
AARRRRG So not only was I using the wrong primers, the right primers NEVER helped because they wouldn’t work on the DNA cause of a LARGE mutation there!
Someone owes us for 2 uL of Sal 1 restriction buffer. But I think that’ll help.
Hehe, personally I LOVE cellular virology. Doing tissue culture work, primary tissue work, growing virus and all that other fun stuff. I can do a little bit of everything except for westerns. YAY! No western blots of me!
And I swear ALL science is like Voodoo. You’ll have 9 months of EVERYTHING working the first time. Then 2 months of everything but the gaslines exploding in the lab.
Particlewill: There’s absolutly nothing to worry about bacterium with viral plasmids in them. The plasmids are made so the bacteria won’t express the viral DNA.
Hardygrrl: Hehe
Gobear: RT can be both of them Generally RT should be for reverse transcription and used as protein/kit names. Reverse Transcription and Reverse Transcriptase can more or less be used interchangably. “Using RT we generated the dDNA”. Personally I’ve never been able to keep in vitro and in vivo straight
Cards: Hehe!
Haha…I had to use a homemade electroporator when i was in grad school…one wrong move, and I’d be dead (or close).
Well, another lab (henceforth know as the IL, or Incompetent Lab), asked us to help with haplotyping a mouse SNP between a BL6 and 129 cross, and since i was bored, i took the job. I run their DNA, all looks good, except there is no SNP where it is supposed to be, but there is one 3 basepairs (bp) downstream. their PI says don’t worry about it, that DNA was a mongrel, and gives me the two controls to sequence. They come up the same at the supposed SNP site, but have different bases 3 bp downstream, where the SNP i told them about was. Turns out they had the location wrong, but they only checked after i had to get my PI to talk to them because i knew they were wrong. Then we finally have the correct position of the SNP, and i reorder correct primers. we run 35 samples of a mix between the two mice populations in haplotyping, and they all come out as BL6’s. WTF? i say, and redo it, with the same results. Then i sequence them to be sure, and all 35 are homozygous BL6’s. IL calls up the people in their lab that breeded the mice. Turns out they breeded the wrong mice. 1 1/2 years of data is ruined. IL’s boss is furious (normally he is a jerk, but now he is mega-ultra-super jerk with cherries on top). a month later, they have 8 new samples from a new cross. I run them, all of them are BL6 again. Yep, their breeders screwed up again!!! Another month later, we get 10 more samples, and finally they get it right. All the while, the guy from the other lab i am helping is asking if we have openings (he would be about number 50 in line to get a job here, EVERYONE on the floor has asked because we are the cool lab)
Such fun, but i got to go back to staring at DNA traces looking for SNPs!
You remind me why I don’t do that anymore. It’s always a “hail mary” pass that might work, or might not, and there’s 2 months down the drain. At least I know what you’re talking about. Thowing that verbage around sure impresses my new co-workers. (BONUS)
Whenever I couldn’t get my plasmids to grow, I first tried again the same way, then tried fresh plates, and then tried fresh bacteria. Then, I would try babysitting the damn things and basting them in positive energy while watching them incubate. Just like talking to plants. It sometimes worked. Good luck.
Apricot: I’m thinking of using radioactive P32 
Wait a minute, am I the only who read the fact that he has powdered steak? I want some powdered steak. Shoot that, along with Tang and I could drink all my meals. 
Amp, you haven’t lived until you’ve had beef flavored sneezes.
Shhhhh! Don’t say anything to upset the mad scientist with access to vast stores of HIV!
Hey, look on the bright side! Your beefy sneezes probably cancel out the monkey-fresh environment at your place of work.
It’s nice to know that if the world ever faces viral Armageddon, CRorex might be the one to tell us about it and we’ll get a few good belly laughs in before we flee to our hermetically-sealed secret bunkers in the Northwest Territories.
What, you guys don’t have bunkers? Well, you sorry flesh puppets aren’t getting into mine!
I could re-fill toner cartridges with powdered steak and print BEEF-O-GRAMS all day long!
Bactotrypone–It’s what’s for dinner!
Congratts CRorex these boards had been making too much sense since justhink was unmasked.
You only think they’re secret. When you get here, you’re gonna have to argue with Charlie Peter Charlie and his seventeen kids, 'cause you built it on his trapline. He’s been using it as a line shack for the last while.
He does say that you’re almost out of sugar and canned goods, please send more. Oh, and try not to mind the drying pelts in the bathroom, eh?
Glycerol? Plasmid?
You IDIOT. You forgot the eye of newt! And was there lightning? NO! Now GET OUT there and find me a newt, you pathetic nematode. And find a living newt, you understand, ALIVE. There should be a lovely storm tonight, so if you’re late, you miserable crawling wretch, I’ll THRASH YOU again!!! Begone! I’ve much WORK to do!
That’s for damn sure. We’ve begun sacrificing chickens to the PCR gods before beginning our tests. I think someone’s sneaking in in the middle of the night and spitting in all our primer stocks once a month or so.
But at least all I have to deal with is human blood. And the occasional chunk of tissue. We got in a whole testicle a while back, but that’s another story.
Oh, and who decided blood is the best source of DNA, when hemoglobin is a PCR inhibitor?!