When Do You Think You'll Be Done?

15 Weeks.  That's how long livejournal says its been since my last entry.  I wish I could say that I've been doing lots of great experiments in that time, but the fact is, I've been trouble-shooting one particular experiment.  See, I used to grow my yeast in a 30 degree room on a shaker.  Then, the Powers That Be decided that room should be an office.  So, they tore out the warm room and I had to start growing my yeast in a shaking incubator.  That's when the trouble began.  Once that happened, my experiment didn't work properly.  It's taken me 3 months to get the damn thing working to about 50% efficiency and that looks like it's as good as I'm going to get it.  I'm going forward with it now, but it ain't pretty.

In the meantime, I haven't done much of note until two weekends ago when I went to Iowa for nephew Benjamin's first birthday and the Iowa State Fair.  Would love to drag John to the state fair one of these years.  The day after I got back to Chicago from Iowa, I headed out to California to see John and that's where I am right now.  Mostly what I've done here is relax.  John and I went to see The Dark Knight and went out to eat to celebrate his birthday (a month late but it's the first time we've been together since his birthday).  I had already seen the movie, but it was fun to see it again.  I got John the straight-to-DVD Stargate SG-1 movie "Continuum," and we watched it last night.  It was fun--sort of like a really long episode.  Otherwise we've just been hanging out which is what I've desperately needed to do lately.

Tomorrow I go back to Chicago and if I'm not exactly thrilled with the prospect, I'm at least not completely dreading it now that I have that experiment working "good enough."

Earlier this week, I had a thesis committee meeting.  Not the penultimate meeting--the one where they say, yes, you're done schedule your defense--just a boring old meeting.  I'm hoping to have the penultimate one in a few months or so but my program requires we have a committee meeting every year and since it had been 16 months since my last one, I had to schedule one now.

It went well.  They liked my data so far, they think I have a good story to tell and that it will be publishable and make a Meaningful Contribution To The Field.  They agree that I will be ready for my penultimate in a few months.

When I got back to lab, my friend and labmate, Rita, asked how it went and somehow she twisted, "Yeah, they think I should be able to have my penultimate committee meeting in a few months," into something spectacular and wonderful and exciting.  Don't you think it's exciting?? she wanted to know.

No.  Now, if my committee had said to me something like, "You know, we think you've done enough.  Why don't you just write up what you've got, schedule your defense for next week and get the hell out of here," THAT would have been exciting.  But that's not going to happen.  We all agree I need to finish these experiments before I can defend and leave.  Getting them to agree to that is NOT the rate-limiting step.  Actually getting the experiments done is the rate-limiting step.  And I have many long, long days in the lab before I am done with my experiments.

The good news is that I continue to make progress and haven't been having the vast amount of technical difficulties I've had in the past.  If I don't pass out from sheer exhaustion, this should be doable.

Well, according to livejournal, my last update was 9 weeks ago.  That's a bit longer than I had intended, but they have been pretty profitable weeks.  One of the many things that has happened is that my paper outline has changed significantly.  The old paper outline has been completely discarded.  This might seem like a bad thing, especially since some of the things I had been working really hard to  do are not in the new paper outline (remember the green dots?  Gone).  But, really, it's a good thing because this paper is better and stronger than the original version.

This paper consists of 5 figures.* 

Figure 1 A has three panels of images.  I have two of the three yeast strains I need for this figure and one panel of images done.

Figure 1B has one panel of images and I just got the yeast strain I need for that.  Hopefully, I'll be able to do an experiment by the end of the week.

Figure 1C is a particular type of experiment called a western blot and I will be using the yeast strains from the above two figures for that.  The experiment itself takes around 3 or 4 days.

Figure 2 is my favorite figure.  All of  the experiments are being done by another grad student.  Hooray for figure 2!

Figure 3A has five panels of images.  I  have all of the yeast strains I need for that one but none of the experiments are done.  It'll take a solid two weeks to get all of the experiments done and all of the pictures taken and images processed and so on.

Figure 3B has 3 panels of images.  I have all of the strains and one of the panels of images is done.

Figure 4 is a very different kind of experiment.  I've done this type of experiment before and I know what the results will be but I didn't have them in a form that was publishable.  We are getting a new instrument to do these experiments and it should be here early in April (which probably means the end of April, but  I have plenty to do until then).

Figure 5 is the Big Unknown.  It consists of a single set of experiments, but I don't have the yeast strain for it, I don't have the DNA to make the yeast strain and I have no idea what the answer will be when I finish the experiment (well, I know what I hope the answer will be but that's not the same thing).  However, no matter what the answer is, the result is publishable so that's all that really matters to me.


*It occurs to me that you might not understand what I mean when I talk about a scientific paper having figures.  Here is a link that should take you to a site where you can download a pdf of a paper (click on view file).  It also happens to be the previous paper published about my protein.  The link will expire after 30 days so if you are reading this more than 30 days after this entry was posted, let me know and I can send you a new link.

So, today, I looked at my yeast under the microscope and I learned that:

1.  One out of 23 of the colonies of yeast that grew up actually had the DNA incorporated correctly.

2.  My original hypothesis (the one that was called into question by some previous experiments) is probably right.

These are preliminary results which means that the experiment hasn't been done so rigorously that I can say with absolute certainty that my hypothesis is correct, but it looks promising!  Which is good news (something that I'm not really used to)!

So, now I need to do the experiment in a rigorous way with all of the proper controls and so forth so I can be absolutely certain of the result.  Due to the nature of the experiment and the fact that I'm going to California soon, I may not be able to do that for a couple weeks. :(  I may be able to do another "quick and dirty" experiment before I leave that will give me a little more information, but I will still have to do a "slow and clean" version later (actually, the quick and dirty experiment is not particularly quick).  The problem is that I have identified the strain of yeast that I want, but now I have to "purify" it by spreading it on a plate and make a preculture (the 5ml [or 1 teaspoon] culture I was talking about the other day) which will take two more days.  The experiment itself requires me to grow the yeast in 10mls (2 teaspoons!) of special media (liquid) overnight, then switch to a different media and grow for 8 more hours.  Then, I have to do 6 more hours of work with them.  I can split it up and do only 3 hours of the work and leave sitting overnight then come in the next day and finish it (which is what I will do since a 14 hour workday is really not fun).  So, to add it all up, that's 2 days to get the yeast ready and three days to do the experiment.  And, I'm leaving for California in the early afternoon on Friday.  Not enough time.  But, if I skip the part where I purify the yeast and do the rest of it, I can get it done before I leave (I'll still be purifying the yeast so that I can do the clean experiment when I get back).

It may seem a bit silly to spend three days doing work that I know I will have to repeat.  But, I can still get some valuable information from the experiment and it would be nice to have it before I leave.

Of course, if some problem comes up that throws everything into confusion again, that would be a really depressing way to start my stay in California.

Sorbitol is not Splenda.  Splenda is something called sucralose which is a chlorinated version of sucrose (sucrose is regular granulated table sugar; sucralose has one OH group substituted with chlorine) while sorbitol is a different sugar substitute used in cough syrups and sugar-free gums.

I am just spreading misinformation all over the place.

5mls really is 1 teaspoon! 

It just looks like so much more than that in the pipet or the tube.  You'll be happy to know that I didn't actually take my measuring spoons to lab.  We have some plastic spoons in the office (for lunch) and I took one of those and put 5mls of water in it, then I took the spoon home and filled it with water then dumped that into my measuring spoon.  It is not a particularly precise way of doing things, but that's okay.

Remember when I got that weird result a couple of weeks ago?  Well, I've been working on making a new strain of yeast that I can do an alternative experiment in it.  I thought I had it, but I was wrong.  So, I had to go through the whole process of making the DNA and putting into yeast all over again.* Today, I'm going to go into lab and see if any of the yeast grew up.  If they did, I'm going to do a quick and dirty experiment to make sure that the DNA went into the right place.  This experiment has the advantage of also letting me know if the results from the final experiment are going to be what I hope they will be.  So, if all goes well, i should have the answer in a few days (of course, you know, it hardly ever goes well, but for some reason I'm still optimistic that it will).  Which is good because I'm leaving for California on Friday and it would be great if I knew what the answer to this question is before I leave.


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*How that works is like this:

1. I do all of the stuff to make my custom DNA (maybe one day, I'll go through how I do that, but it's pretty complicated).
2. I grow some yeast in a liquid culture.  A culture is just what we call a sample of yeast, or bacteria.  The liquid is called media and it's made of nutrients and sugar so the yeast have all they need to grow happily.  First, I grow a really dense small cultures (about 5 milliliters (ml) which is probably equivalent to something like 3 tablespoons [aside:  I looked up a converter online to see how much it would really be and they are trying to tell me that 5ml is 1 teaspoon and I just don't buy it.  I think I may take my measuring spoons into lab.] [edit:  I was wrong, 5ml is 1 teaspoon) in a tube.  Then, I put a drop or two of that into 50 ml of media (in a flask) and grow it overnight at 30 degees Celsius (86 degrees F) (because that's the temperature yeast really like to grow at), on a shaker which swirls the media around which makes sure that lots of air gets to the yeast because yeast need air to live.
3. When the culture is at the right density of yeast, I add two chemicals, and incubate it for a little while longer.  These chemicals help chew away at the cell wall (yeast have a cell wall, like plants do) which is necessary in order for them to take in the DNA.
4. Then, I spin the culture in a centrifuge and all of the yeast goes to the bottom of the tube (this is called the pellet) and I dump out the liquid (called the supernatant or just sup [pronounced like "soup"].  Then, I mix up the yeast with water and spin them down again--this makes sure all of the media and nasty chemicals are gone.  Then, I mix them up in a solution of sorbitol (which is what Splenda is made of--but we don't buy a big thing of Splenda or anything like that, we have a big tub of sorbitol from the chemical company [edit:  oops, sorbitol is not Splenda after all]) which is just a nice, cushy liquid for them to be in so they are pretty happy.
5. Then, I add my custom DNA to the yeast and shock the hell out of them.  Literally.  I put a little bit of the yeast plus DNA in a tube and put it in a machine that zaps them with electricity.  This makes the yeast bring into the cell anything surrounding them which includes my DNA.
6. Then, I put the yeast on an agar plate and let them grow in a 30 degree incubator for two days.  They grow up in little round patches called colonies.  Now, the way I know that my yeast took up the DNA is that this is all set up so that the only way they can survive and grow is if they took the DNA into the cell.  For my experiments, I need them to take up the DNA and paste it into the DNA they already have and it needs to be pasted into the right spot.  Because of the way the DNA is designed, it should preferentially paste it into the right place, but oftentimes it doesn't.  So, then I have to check if the DNA got pasted into the right spot.

Total time for this procedure:  2 days to grow the small culture (but I only use a little of it, and I can keep the rest in the fridge for the next time I need it), 1 day to grow the big culture, about 1 hour for the spinning and adding DNA and zapping, then 2 days for the yeast to grow up on the agar plate then another couple of days to see if the yeast pasted the DNA in the right place, then 4 more days to purify the yeast to make sure I have a pure strain and every single yeast cell is exactly like all of the others, then 2 (very long) days to do the real experiment.  If everything goes right.  If it doesn't, it takes much longer, of course.  At this point, I should mention that yeast is considered a very fast system to work with.  It takes much, much, much longer (several months, and that's only if it all goes well) to put a piece of custom DNA into a mouse, for example.  It takes two hours for the yeast to reproduce (divide into two, essentially).  It takes mice a few weeks to grow up enough to be able to reproduce, then you have to mate them and I think it takes something like 20-30 days for them to have the babies (pups) then a few weeks for all of those to grow up enough to be able to reproduce and you have to do that a few times in order to get your mouse that has the right DNA in it.  And they're expensive, too, because you have to keep them in a special facility and you have to pay the facility to take care of them.  We store yeast in the freezer or cold room, or on our bench and don't have to pay anyone to look after them.

Step 1:  Survey your apartment looking for beverages that are both festive and breakfast-y

Step 2:  Assemble your ingredients

• Orange Juice
• Cranberry Apple Wine
• Sparkling Water
• Various Spices (cinnamon, nutmeg, and ginger
  

Step 3:  Combine equal parts of each liquid (1/3 cup in my case), and sprinkle in spices (a couple of dashes of each for me).

Step 4:  Chill

Step 5:  Serve with eggs and sausage and pastries.  Yum!

Have a merry Christmas everyone!

Well, I'm back in lab, doing experiments, going to lab meeting, and all that good stuff.  This week, I'm trying to get everything ready to do the experiment that's the alternative to Experiment 2.  First, I had to put some custom DNA #1 into the yeast.  Then, I had to make sure that DNA #1 was in there like I thought it was.  Well, then I found out that although it was in the yeast, it did something a little funky and that's when I remembered some experiments from way long time ago and thought, "Hey, did this same thing happen back then?"  And then I panicked and got nauseous and started beating myself up for not doing the control experiment that would have made it all okay.  But, then I sat down and thought about it more and realized that it was all going to be okay and I wasn't completely screwed (although I was still an idiot for not doing the control experiment).  Then, I made my confession to my advisor who also thought that it would probably be okay, but first we had to figure out exactly what had happened to the DNA when I put it into the yeast.  So I've been doing that. 

Remember, this isn't a "real" experiment--it's the equivalent of chopping carrots to put into a salad.

Anyhoo, now I know what happened to custom DNA #1 when it went into the yeast so now I need to put in custom DNA #2 and then when I have that all done, I can do the experiment.  So, I should be finished with putting in DNA #2 and have the yeast all ready by Christmas Eve or so, if everything goes the way it's supposed to (which it won't, because it never does) I will be able to do this experiment at the end of next week sometime.  And then we will know if my hypothesis is correct.  Hopefully, it will be.

I haven't started any of the books I got for Christmas yet.  I might be able to start on Christmas Day.  Maybe.  I'm not going to lab that day, anyway.  I'll be in lab everyday between now and then, but not on Christmas.  Sometimes, you just have to take a day off.

It's raining cats and dogs here in Chicago, and has been all day.  Can't wait until later tonight when it all freezes and turns the city into a giant skating rink!  Perhaps I should bring my ice skates home from lab so that I can use them to get to campus in the morning.

Remember the experiment that wasn't?  Well, I finally got everything together to do it.  It was a chore and a half, let me tell you.  Every step of the way I ran into problems.  There was one point where I thought I was actually going to cry from frustration!  Anyway, the good news is that it worked.  The bad news is, it was a "gauge swatch" kind of experiment.  It was an, "Is this working okay?  Because if not, I've got to start all over but if it is okay, then I can move forward and do something cool."  So far, it looks okay, but I've got another test going right now to make sure.

And people wonder why we still haven't found the cure for cancer!  Sheesh!

Ghiradelli story: 
Last night when I got home (at 11PM!), Ghiradelli ran out into the hall as he normally does when I come home.  As usual, I held the door open with my foot and put my coat away.  I was about to go out and get him when he comes racing back into the apartment, ears back, tail down, trying to make himself as low as possible and goes around the corner into the bedroom!  I look out in the hall and at first I see nothing, but then I see a guy come around the corner with his dog.  There hadn't been any barking so I don't think the dog actually saw Ghiradelli.  Poor kitty, he didn't come out of the bedroom until I had shut and locked the door, then he peeked around the corner to see if it was okay.