Fluorometer angst

As I predicted, I haven't gotten as much done this week as I would have liked but overall I have made progress. I successfully taught Cho how to electroporate bacteria and plate them on Amp-LB plates. He did a good job. Today I will teach him how to inoculate growth media so that tomorrow we can isolate his DNA.

A couple of months ago I bought an instument called a fluorometer. It uses fluorescent tags to measure protein, RNA, and DNA. Most fluorometers are very expensive ($10,000-$20,000) but this one is a simple unit that measures one tube at a time. It seemed to meet our needs perfectly so I spent the $700 our sales rep asked for. Sure enough, the Qubit (that's the model name) is easy to use but I have been having second thoughts about its accuracy. After measuring protein levels, I adjust each individual sample so that I am loading the same amount of protein in each lane on my gel. However, when I go back to the gel and stain it with silver or with coomassie blue, I am finding that there are different protein levels. As a notoriously poor mathmatician, I went back over my number to make sure I calculated everthing correctly - I did. This means that my raw data is incorrect. I wonder if there is anything in the cell lysates that autofluoresces? You would think not. If so, that is a problem that should have been addressed long ago by the many instrument manufacturers. I ran a protein assay again yesterday and am going to run four gels today. The first two I will stain with coomassie blue to double check my protein loading. If my results mirror my calculated data, I will rerun the gels this afternoon and prepare them for Western blotting. If they don't agree with my calculated data, I will have to make the necessary adjustments by visually gauging how much to load. Then I can rerun them. Enough writing. I have a lot to do.

I get paid for this? How cool is that?

Another Monday. I have a lot to do this week but realistically, I will probably only get to about 50% of it. Part of this afternoon is already shot because Michelle and I are refinancing our house so I have to go to the bank to sign papers. Here's hoping that I get more done this week than last. I got sick last week and ended up taking about 2 days off. Luckily, the university gives us great benefits so I have a lot of sick time (about 30 days accrued) and a lot of vacation time. Even with my recent trip to London, I will have maxed out my accrued vacation time. The university gives me 16 hours of vacation a month. Additionally, I elect to trade 12 hours of monthly sick leave for an additional 4 hours of vacation. The University of Iowa allows us to accrue up to 480 hours of vacation time. I have hit that mark and will not be able to recieve any additional time. I am going to have to take some more vacation. Darn. Now that it is warming up, I will probably take a couple of days off to get my flower beds ready and planted. I find that to be very therapeutic.

Anyway, I have a couple of tissues to process for protein today. I will probably run the gels tomorrow. Also, tomorrow I will be teaching one of our research fellows how to extract DNA from a filter, get the DNA into bacteria, and grow the bacteria on a selective plate. If we are successful, then I will teach him how to amplify the colonies, extract the inserted DNA, and sequence his insert. Hopefully this will go smoothly. I just hope this guy doesn't expect me to do it for him. My job is to teach him how to be independant. Unfortunately, for many of these surgeons, it is easier for them to rely on someone else since they have a relatively limited amount of time in the lab (usually 1-2 years). However, if they want to be successful researchers, they at least need to know the principles behind the science and have a working knowledge of the techniques.

What I am perhaps most interested in this week is doing the phospho-merlin Western blots of the tissue I collected before a couple of weeks ago. The last Western gave me inconclusive results. Hopefully this one will be a little more convincing.

Oh, we also just got a paper accepted for publication. It is a project that I worked long and hard on. As a preliminary study, it is pretty good but there is much more that can be done with it. The only drawback is that it is labor intensive - the mice require daily treatments. Oh well, that is what I am here for.

I like to be busy

This afternoon just flew by. I spent this morning getting caught up on email and other things that come with being gone for a week. This afternoon was spent isolating protein from tissue samples. I had seventeen samples and after about 3.5 hours I finally finished. Tomorrow I will start running gels again.

More surgery

The sutures I made last week on my rats that I thought were dehiscing actually didn't. Well, one of them did partially but the others actually looked pretty good. No infection either. Today I performed the second part of the experiment. It only took me two hours to implant the pumps and catheters in the four rats. Not bad. Most of the rats went under very quickly but one rat just wouldn't give up. I had to inject her three times with ketamine. While they were under, I also injected them with an analog of uridine (a nucleic acid) called BrdU. When cells divide, this compound is taken up during the DNA replication phase (the S-phase). We have antibodies that recognize BrdU and will therefore allow us to get an idea of which cells are dividing. Since BrdU has a short half-life, I will do two more injections tomorrow before I sacrifice the animals and harvest the nerve samples. I am anxious to see the results of this experiment. Our hypothesis is that we will see an increase in phosphorylated Merlin in the nerve section distal to the cut. We also believe that we will see an increase in cell proliferation distal to the cut.

My morning is blown.

I really wish something would just work. My job is a lot like my golf game. I am a lousy golfer but every now and again, I pull off a tremendous shot. That one shot out of 100 (my brothers may remember that 321 yard drive straight down the fairway on the first hole we played right before Adam's wedding) is all it takes to bring me back. Research is a lot like that. Most of the time, we fail but it only takes one good result to keep us going. I've been swinging a lot lately but just haven't connected.

Last night I innoculated three flasks of growth media with bacteria and not one of them grew. This would either indicate that my bacterial stocks have gone bad (unlikely) or that I am using the wrong antibiotic (also unlikely, but I went by memory so I could be wrong). This process is about as basic as washing dishes so not a lot should go wrong. I am going to check my notes to ensure that I used the correct antibiotic.

This particular project has been a real pain in the neck. We got these three DNA constructs (A DNA construct is generally in the form of plasmid or circular DNA. Your gene of interest is inserted into a larger chunk of DNA that contains sequences to enhance the production of your gene of interest such as a promoter and a poly A tail. The plasmid backbone also contains a gene that for antibiotic resistance to assist in the selection of bacterial clones that contain your gene of interest) from another researcher but didn't get much information about them. Generally, when you send someone a plasmid, you also send them a plasmid map and a sequence. We got nothing. I didn't even know what plasmid backbone my gene was in. I had to design some DNA primer so that I could sequence my gene from the inside out so that I could see what the DNA sequence upstream and downstream of my gene was. I then took this data and ran it through a data base at the NIH (a process called BLAST). The results indicated that my gene was in a plasmid called pCDNA 3.1-. pCDNA 3.1- and its family members contain an ampicillin resistance gene. The typical way to store and mass produce this plasmid DNA is to insert it into E. coli bacteria. It is stored at -80 degrees C and when needed, a small stab of frozen bacteria is placed into growth media and incubated overnight with the appropriate antibiotics. Unfortunately, my bacteria didn't grow last night. Back to square one.