That’s great news, Ogre. Between that trip and working on quals, I think you’ll have everything you need to get your head and your experiments back on the right track.
I often feel like bench science is like being in an bad relationship. As soon as you are really ready to quit, literally walking out the door, it shows up with flowers and you are head over heels again. I’ve been doing that dance for almost 13 years.
Wow - loads of Yay!
I’m not going to advise leaving the program. It appears, based on what you’ve written, that this is something you want to do and you can picture yourself being happy with a job in this field. I do want to say, speaking as a dropout from a doctoral program, you do not need to feel guilty if you choose to leave. The university makes decisions about admissions, funding, and everything else based on the knowledge that some students will take off. Further, it seems you’ve figured out that some of the professors don’t exactly have your best interests at heart.
There’s one of those in every department.
The extent of my knowledge about plants is limited to the ‘experimenting’ I did in high school in between classes.
However, I just read through this thread and let out an almost-audible whoop when I read this good news.
Wishing you all the best,
mmm
I thought I’d give an update.
I put my experiment aside for a couple of months so I could concentrate on my written qualifiers. I passed them with pretty much flying colors. None of my profs had any major issues with my answers, and I got an unqualified “pass” out of all of them. That’s the good news.
The bad news is that my plants are still unrelentingly recalcitrant. I didn’t go to Gainesville. I went to Cornell University to work with the other authors of the book. After a week of throwing every trick they knew at my plants, no DNA. They were extremely kind and helpful, and felt awful that they couldn’t help me. Just my damn bad luck that I happened to pick such a tough nut to crack.
I still have some ideas, but they’re quickly getting thin. Today, I ran another extraction with fresh tissue, and nada.
Sigh. This hurts me in places I didn’t know I had. I have my oral quals in two weeks, and the major part of them will be talking about my project. This, obviously, is a big problem. I don’t want to have to contemplate a personal and professional failure on this scale.
It’s beginning to look as if I wasn’t overdramatizing in my OP. My funding may run out, and I may really never get my PhD.
Now, if you’ll excuse me, I’m going to go cry.
Hi Ogre. I don’t have any advice or suggestions, but I wish I could help in some way. Sending support and hope your way.
Really dumb sort of question about the degree program.
Ok, from what I sort of understand you have to have some sort of project that you discuss and perform at great length [and repeat lots and lots of times] right?
So, isn’t a negative result still a result? You proved that this particular theory is not correct, and you can document at great length why it doesn’t work.
Isn’t that still a contribution to knowledge? Can’t you base your thesis on theory X and why it is incorrect?
I’m saying this as a faculty member who has mentored numerous PhD students.
PICK A DIFFERENT STUDY SYSTEM.
I am amazed your advisor let you waste this much time on this (actually, based on your description I’m not).
DNA extraction is usually dead easy. If you can’t do it on your plants, I am way inclined to think it is the plant, not you. Go pick a different species that doesn’t have this problem.
Unfortunately not. It’s not that I have a negative result. I have NO result. You can’t answer DNA-sequence-based questions if you can’t get DNA. And I have been spectacularly unsuccessful at getting DNA.
Thank you very much. Yes, I should have picked a different system a while ago. I was “led on” by the fact that the authors of the paper about the phylogeny of the taxa at large (not including my species) told me that they had no problems at all getting good DNA. The congeners the worked with are all xeric-adapted as well, so I thought, “Hey, it has to be me, then.”
Also, unfortunately, because I have invested so much time in it, it’s going to be very, very difficult to start over. I have little time left, and I’d have to have at least another field season to sample, and the next growing season is months away. Rock, hard place.
You are not going to finish on time. Accept that first, so you can move on to the important stuff. You have 2 choices:
keep doing your current project. if you do this, chances are you won; finish at all.
pick a new species, one where you know you can extract what you need. Put in an extra year for fieldwork/labwork. it’ll take longer, but it is the only way you are going to finish.
You could try finding a collaborator that already has samples collected. Assuming there are no nasty congeners in the plant, DNA should be easy to extract from dried samples etc…
Are you doing molecular phylogenetics, population genetics or what?
Both. These are recently-discovered outcrop endemics. I’m trying to do the phylogenetics so I can see where they fall in the clade, and so I can get an idea of the evolutionary history (these plants are very isolated, with the nearest possibly ancestral congener living at least a couple hundred miles away. How they got there, and whether they speciated from that congener or something else altogether, are big parts of my project. I am also trying to do a clock, to get a rough idea of the time of speciation.) Also, since they are isolated in small patches, with small population size, I want to do the population genetics to get an idea of allele fixation, migration rates, etc.
What marker? I assume mitochondrial DNA?
If so, you could try isolating the mitochondria BEFORE DNA extraction. There are kits and protocols that will give you very pure mitochondria preps.
e.g. here is one
After you have the pure mitochondria ,then you can do the DNA extraction.
mtDNA is not terribly informative (usually) in plant phylogenetics. The markers I’m trying to use are a mix of chloroplast and nuclear DNA (and AFLP uses whole genomic DNA). I’ve thought about trying to isolate nuclei and chloroplasts on a sucrose gradient, and I may yet try to do that. I’ll give the fresh tissue extraction another go on Monday morning (I may have messed up somewhere yesterday. It was pretty rushed, and I may have used much too aggressive a protocol.) If I still get nothing, I know I’ll have exhausted all reasonable options, short of highly specialized extraction techniques, and I might have to move on. Another problem I’ve had is that I’ve had to raise all of my own money. I’ve done pretty well, but molecular techniques are expensive, and the money’s getting thin too.
To what I’m going to move on, I’m not sure, honestly. It may be too late. These plants (about 10 endemics) do their thing in high summer, and they’re mostly gone now. I was frankly floored yesterday when I found my plant still alive out there. I thought I had a new lease on life. I guess I still might. We’ll see.
Ah. Serious bummage. I always got the impression that DNA from reasonably fresh plants was fairly easy to extract, after all my goddaughters did that extract teh DNA from an onion thing in girl scouts.
Can you work with dried matter? Can you actually get dried matter?
/me tilts head like puzzled puppy … never did lab work but it sounds interesting.
good luck!
Theoretically, you can get DNA out of any plant. Onions are relatively easy for a few reasons, but the biggie is that they are polyploid (meaning they have multiple copies of each chromosome in each cell, and thus, have tons of DNA to extract. Also, when kids do DNA extraction on onions, it’s usually not intended for any further use, and that wad of stuff in the tube is not really pure DNA; it’s a complex of DNA and (probably) lots of polysaccharides.
That’s actually the main thing that has cost me soooooooo much time. I really didn’t know better when I started this, and I had read several papers in which DNA was extracted from freeze-dried material. I adopted that technique, because I had TONS of samples, and needed a way to preserve them more or less indefinitely, so that I could get to them all.
Big mistake. Apparently, in plants with lots of aromatics (check), polysaccharides (check), and polyphenols (check), freeze-drying is strongly contraindicated. Upon freeze-drying, all those nasty organics tend to complex with the DNA, and then co-purify upon extraction. In other words, you end up with a nice, big pellet of “DNA”, but it’s useless for further analyses.
It really sounds more complicated than it is. The idea with the sucrose gradient is to isolate the chloroplasts (containing chloroplast DNA) and the nuclei (containing nuclear DNA) such that you don’t break them open and expose the DNA to assault from all the previously-mentioned nasties in the cell.
Thanks! My wife told me yesterday that she said a little prayer for me, asking God to allow me a little success and encouragement. I suppose he wasn’t listening.
Well, keep soldiering along, at least you know what the failure point is, and can correct it. Knowing and being able to explain where the fail is without getting seriously whiney or excuse laden can be a plus.
Also a molecular biologist with a bit of plant background. Dr Mrs Slayer is also and a doper too.
Maybe be set up a new thread or blog and take us through the experiments. When I worked with algae we could do no molecular biology unless the nucleic acids were extracted with CTAB - cetyltrimetylammonium bromide - to remove sulfated proteoglycans etc
WRONG.
Well, as far as your PhD is concerned, but you don’t think that would score some points with a potential employer?
Damn, if you were in my field, and had had similar experience and demonstrated that sort of ability to teach yourself complex concepts, I’d be hounding you to get your resume into USAJOBS yesterday. I’d want you working for me right away, because before long you’d be able to do my job.
Unfortunately for me, I’m in statistics, not biology. But there have to be people out there who are looking for someone with the skills and aptitudes you’ve demonstrated in your quest for the doctorate, and care more about that than the credential itself.
Sorry the experiment didn’t work, but isn’t science also failed experiments? Why did it fail? Write it up well.