We’ve had a pretty good relationship over the years. So, why are you being so stubborn these days? In particular, why have you decided to build a cell wall that cannot be permeablized with lyticase and beta-mercaptoethanol (but only when you are in a particular growth media)? This behavior is really unacceptable and it is actually going to get me into trouble with my committee. I was supposed to have my penultimate committee meeting this month and because I can’t get the IF pictures and subsequent quantitation (because of your little cell wall issues), I’m not going to be able to have it.
I am especially puzzled because everything was fine between us before and only in recent weeks have you had these cell wall issues. Perhaps you are angry about the whole killing you for science thing. Perhaps you think I will stop killing yeast if I can’t get you to behave. I assure you that is not the case. I will keep trouble-shooting this experiment, killing millions of yeast in the process, until it works. This is not a threat, dear yeast, just reality. But, if you cooperate with me, then your death will not be in vain. I will be able to get information that will be useful to scientists all over the world and I will put your picture in a nice journal, maybe JCB or even better. Won’t that be better than dying a silent, unnoticed-by-everyone-except-me-and-my-advisor death?
Perhaps you are foiling this experiment because you realize that I will not be able to leave if I don’t have it and you don’t want me to go. I appreciate the sentiment, dear yeast, and we’ve had some good times over the years, but I really need to finish and graduate. And it is not like you will be lonely–far from it! There are many people in the lab who will still be doing experiments with you long after I leave. And they will all being trying to make you famous, just like I am. I promise.
So please, please, please, dear yeast, let me go. Lower your wall and let the antibodies inside so that I can take beautiful pictures of you and write the paper and my thesis and go to California and live with my husband and make babies.
Sometimes, a search engine term shows up in my stats that I feel I should address. Today, it’s this:
ypd media does it ever go bad
No. Well, it does get contaminated (if it’s cloudy, it looks contaminated). Then, you need to get rid of it. But, in general, I have used media that is a few years old and the cells grew fine (I was desperate and I found some that belonged to some former student). Beyond that, I suppose I couldn’t say, but usually people use their media within a year or so of making it. If you don’t, then you are likely making too much at once which I wouldn’t recommend because of the risk of it becoming contaminated before you get around to using it.
At least, that’s what I assume you mean when you ask if it goes bad. If you are talking about how it gets darker after autoclaving, then I would say that is normal–it’s just the sugar carmelizing. If you leave it in the autoclave longer than a typical 20 minute cycle, it can get quite dark–almost black. It’s still okay to use, though.
There, that’s my public service for today.
I seem to have lost the month of June. Or rather, I may have misplaced about 20 days of June. I swear to you that only yesterday it was about the 5th or so and now today it is the 23rd. I wouldn’t be so concerned, but I was supposed to have my penultimate committee meeting in June and it doesn’t look like that’s going to happen because I still have a few key results I need before I can have the damn thing.
So, if you see a few days of June just laying around, could you send them over to me? Thanks.
Step 1: Purchase silver polish.
Step 2: Take silver polish to lab.
Step 3: Load gel and start it.
Step 4: Put primary antibody on IF.
Step 5: Grab a velvet (commonly used for replica plating yeast; the velvet doesn’t have to be sterile, but generally the only clean velvets in our lab are sterile).
Step 6: Put on gloves (latex or nitrile, your choice) squirt some water onto the velvet using your water bottle to dampen it.
Step 7: Squirt silver polish onto velvet. Use velvet to smear polish all over jewelry. It’s okay to get it on the stone if there is one (well, it’s okay to get it on malachite anyway).
Step 8: Let silver polish dry
Step 9: Image gel. Realize PCR didn’t work. Cuss a blue streak.
Step 10: Use water bottle to dampen rest of velvet and wipe off all silver polish. Wet a cotton swab to use in the nooks and crannies (though I like to leave a little tarnish in embossed areas because it emphasizes the design).
Step 11: Do a final rinse of jewelry using water bottle. Dry with paper towel. Let dry.
Step 12: Rinse IF and put secondary antibody on.
Step 13: Put jewelry on.
Because there were two copies of Physics Today on top of the mailboxes and neither one of them belonged to my husband.
I think I will make sure to subscribe to Cell or something to make sure I don’t drown in physical sciences when I live there permanently.
In other news, my husband had to get an MRI for the severe headaches he’s been getting (probably migraines). He was extremely nervous about it having already had to cancel one appointment due to panic attacks (he’s never had panic attacks before). So, naturally, when we go to the office to get the test done, he doesn’t bring a thing to do while we wait. Finally, I made up several geometry problems and and the following word problem:
What’s the smallest number of children would we have to have in order for their combined ages to be 2/3 of our combined ages in 20 years? The ages of the children must be two years apart.
He was happier than a pig in mud doing those math problems. He’s the only person I know who can be calmed down by math.
And now I’m back in GradSchoolTown. Sigh.
P.S. The answer to the problem is slightly over 4 children. So, four children and pregnant with the fifth. We are sooooo not having four or five children. Two will be plenty, thank you very much.
Go read this post over at DrugMonkey (and some of the comments because there are several funny stories in there). When you are done laughing, come back.*
I confess, I have my own secret stash of things in the lab. Not expensive reagents like Taq, but a Backup Supply of things that we routinely run out of (the fact that we routinely run out of critical things is a topic of a whole ‘nother post entirely). In an unlabeled cabinet near my bench I have the following:
- Two boxes of medium-sized nitrile gloves
- Two packages of paper towels
- A box of each kind of tips (this is overly neurotic–we haven’t run out of tips in forever)
- A bottle of LB that I made myself
- A bottle of YPD (yeast media) that I made myself
In the freezer, I have my own tube of ampicillin, and my own tube of ligase buffer (this is for self-preservation; people leave the buffer out for too long and it starts to go bad and then your ligation doesn’t work and it makes me want to howl in fury, so I keep my own buffer since I know where its been; also they supply so much buffer with the enzyme that everyone in lab could have four tubes for themselves at this point).
I’ve also been known to keep a couple of spare bottle-top filters.
I’m pretty sure I’m not alone in this habit of mine. I’m guessing everyone in lab has a secret stash of something or other. What do you guys stash away?
*I laughed at those stories until tears came out of my eyes. Also, if I was the PI, I think the incident would not have happened, because I would have followed PdA back from lab meeting and made damn sure he gave PdB the antibody in question. However, let’s say it happened exactly as written. I would send them home for two days, call them both into my office and tell them in no uncertain terms that if anything like that ever happened again they could both find new labs to work in (this might be somewhat complicated if they have their own fellowships, but no way in hell would I want them in my lab).