Monday, December 17, 2007

Lab Lesson of the Day: Read the god damned recipe

Guys, Im never going to finish grad school.

Stupid Mistake #1
-- Not checking the pH of my solutions.
Stupid Mistake #2-- Not checking for azide in commercial reagents.
Stupid Mistake #3-- Forgetting to put an ingredient in a master mix.

For the past week Ive been trying to optimize this RT assay. I take a few microliters of a viral stock, gently blow up all of the viruses, and measure the reverse transcriptase activity. Its an old, old protocol, should be a matter of copying the recipe someone put in a publication, ploping in my virus, and YAY! RESULTS! Except dot-blots are supposed to look like this:
My dot-blots looked like this:
Yeah... so, Bossman was like "You need to check your reaction buffer. Something is wrong in your master mix." Me "NO WAY! Its right! Its got to be something else!" Bossman "Check your buffers."

*5 minutes later*

Me "Yeah... I didnt add EGTA..."

Reverse transcriptase needs Mg2+ to work. BUT you need the charge of the reaction solution to not be too positive, as that will prevent the reaction product, DNA-RNA hybrids, from binding to a special membrane. EGTA 'neutralizes' Ca2+ ions in the reaction, but leaves the MG2+ ions alone! Super! But not adding it means the reaction buffer blocked the DNA-RNA from binding, so I got these weird twisty halo thingies...

It was a really stupid thing to overlook.

*face palm*

14 comments:

Bill said...

Now you know why analytical chemists (moi) make great cooks.

Forget EGTA in preparing an omelette? Never!

Tyler DiPietro said...

No matter what field you're in, you're going to make mistakes early on. Judging by how quickly your boss appeared to know where the problem was, and how insistent he appeared to be afterwards, it seems as though he's encountered this mistake before. Don't be so hard on yourself!

Brett said...

hey abbie, i liked your idea. I've been trying to find good ideas of things to do on Darwin Day in Feb. Your idea fits well me thinks.

Stacy said...

Probably doesn't make you feel any better, but I dropped a wrench on my well tray of 100 samples and now I need to re-make about 50 of them. stupid clumsy me. :-( But I haven't told my adviser... and if everything goes as planned, he will never know. :-)

SFMatheson said...

HA! I can top that. A few months ago, I joined a lab that uses chemiluminescence and film for blots (blecch; once you go to Odyssey you'll never go back to that medieval madness). My westerns should have looked like this [holds up random page from JBC] but they looked like this [holds up picture of albino cattle in blizzard]. Changed everything, found the problem was...azide in the wash buffer. HA!

Fred Ross said...

Good heavens, if those are the only major experimental mistakes you've made, you're doing really well.

I'm still trying to get things like growing bacteria, making agar, and isolating DNA straight. Remember, progress towards your thesis does not require consistent success, just success often enough to move you forwards at a reasonable pace.

Albatrossity said...

Yeah, when you get to be a PI, you can use those three magic words ("Check your buffers") on your students, and they will think you are brilliant too! We've all done it, and we've all learned from it.

Similarly, I would never never never use anyone else's solutions for an experiment. I did that once, and spent the next week on a wild goose chase trying to repeat what looked like a really interesting experimental result. As it turned out, it was merely an artifact, and due to a different formulation of a buffer solution made by someone else. So you will probably have that experience someday as well, and it will be much more memorable (and hence useful) than this blog comment.

Shaven Yak said...

I maded you a picshur, but i no can has <img> tag.

Bob O'Hara said...

1. shaven yak - that it brilliant
2. In my dark and embarrassing past, I was so good at DNA extraction I could even get it from my negative controls.
3. I use a variant of "Check your buffers" when teaching WinBUGS, but it's slightly longer ("You've given your precision a Normal prior"). Same thing - you get to see the same problem every year.
4. The first month of my PhD, a note appeared in our lab: "Warning, high stress levels! James [my supervisor] working in the lab." You try having a conversation with that dangling from a cord behind him.
5. Ahaha! You've now been struck by a blog meme.

Bob

firemancarl said...

Abbie, can't you just tell 'em that you had a nice convo with Behe and Debmski and you now realize that God and Jebus did it???

Blake Stacey said...

I once made a circuit board explode by trying to measure voltages.

Multiply that by N, and you begin to appreciate why I went into theory. . . .

Ditto on Tyler DiPietro's remark: one can only recognize the problem that well and that rapidly if one has seen it before.

oleg said...

And I tripped the world's biggest cyclotron once...

Che said...

We had a saying in my old lab: "If it works the first time, you have probably done something wrong." Biochemistry/molecular biology is not for the faint-hearted.

Rick said...

Biochemistry/molecular biology is not for the faint-hearted.

Which is why Dumbski writes propoganda now instead of working in his alleged field.