Thursday, February 17, 2005
Stress is making me illI've finally come to what, in hindsight, is a bloody obvious conclusion. Work is making me ill. For months I've been controlled by my insomnia, with no desire to shake myself out of it. For the past few weeks I've had headaches and undefined nausea. Several times I've rang in sick with my headaches and nausea and sleep deprivation making me unable to face getting out of bed. I typically spend all weekend in bed. That then makes me feel more stressed as I feel that I am not getting enough work done. The 3-weekly meetings with SWMNBN seem to loom over me as I face yet another meeting getting chewed out for not having enough results.
Tonight it came to a head. I was ill yesterday, with a splitting headache that left me feeling nauseous. I rang in sick again. Today my headache was improved and I went back into work, making it in in the early afternoon. The day started well. I have been trying to get those blasted southern blots to work. However, I had nice new DNA had made up some fresh solutions and was going to use a new improved technique. I was feeling better than I had in weeks. It didn't last.
The "old" style of southern-blotting is decidedly low-tech. You spread your DNA out by running it through a gel, so that the larger fragments get deposited near the top of the gel and the smaller fragments run toward the bottom. You then soak the gel in 2 different solutions, before covering the gel in a special membrane that the DNA can bind to. You cover the gel and membrane with a stack of paper hand towels (yes you read that correctly) and stick a weight on top. Fill the tank underneath the gel with another solution and leave overnight. Capillary action draws the solution out of the tank, through the gel and into the paper towels. Along the way, the DNA is released from the gel and binds to the membrane which you can then analyse with radioactive probes at a later stage.
As you can imagine, that is a lot of hassle, and the need to blot overnight adds another day to the whole procedure. So instead I decided to use the a vacuum blotter. In this case, a vacuum is applied to the gel so that the DNA moves faster. All the solutions are poured on top of the gel and are pulled rapidly through by the vacuum. It takes about an hour. I set everything up and was good to go. No vacuum pressure. I checked all the tubing - 50 millibars of pressure at every junction as I wanted. But nothing when I connected it to the gel tank. After 2 hours of fiddling and re-reading the blotting machine's manual, I was at a loss.
It got worse. To analyse the membrane I need to make a radioactive probe. This is simply a stretch of DNA matching the gene that I am looking at, created by a simple technique called PCR. To make the probe, a simple reaction is performed that involves replacing the normal Cs in the DNA with radioactive Cs (DNA remember is a stretch of A, C, T and Gs in a specific order). One problem that we may have had in the past is that the PCR reaction that produced the DNA probe may have left behind enzymes and other chemicals that interfere with the making of the radioactive probe. So I decided to purify the DNA first, using one of the kits that I talked about in yesterday's Tuesday Twat. I loaded about 5000 nanogrammes of the DNA (loads) onto the column and cleaned it up. I then checked the DNA concentration of the cleaned up solution to see what volume I would need to use to make the probe. Total DNA left? 100ng. Where the Fuck has the other 4,900 ng of DNA gone?
With not enough DNA to make the probe, and the vacuum blotter refusing to work, everything seemed to come crashing down. The thought of another meeting with SWMNBN with no data to show seemed overwhelming. I kicked my stool across the lab and threw my lab coat on the floor in disgust. Fortunately at 9pm no one else was in the lab (although a few people were still in the write up room). I went for a walk, before locking myself in the toilet for half an hour. I am glad that no one was there to see me as I was swearing uncontrollably and unable to form a coherent sentence. The headache had come back and was making me feel nauseous.
I phoned my parents and to their credit they didn't comment once as I used the word fuck more times than I have ever done in their presence in my life. I have never come so close to simply getting up and walking out of the building never to return.
Well, tommorrow is another day. After regaining my composure I placed the gel in the fridge. I will get anyone who has ever used the vacuum blotter to come and take a look at it. I will make completely fresh solutions and try purifying the DNA probe again. I have until Wednesday to produce a decent southern blot. If I don't, then I don't know what will happen. I will have to try and have a chat with my more sympathetic supervisor before the meeting with SWMNBN. Tonight I considered simply refusing to attend Wednesday's meeting - but that is a hell of a line to cross. I could probably pour my heart out to my doctor and union rep and get myself written off on stress leave - but again there is no going back after that.
I think that I have finally realised that this project was a waste of 2 years of my life. It was poorly conceived from the start, with my role too poorly defined and dependent on other people's success. They haven't produced the goods and have now left, leaving me to pick up the pieces. I took the job because I had no option. My PhD funding had finished 9 months prior and my parents were having to pay my rent for me. I don't think that I am going to get any publications out of it, making it a disaster for my career. My contract is due to expire in July. I have decided that I will start applying for jobs now and explain to the person interviewing me that the project was a waste of time and simply hope that I find someone willing to give me a second chance in a crowded job market. Fuck waiting for July, I'm out of here the moment I get another job. I'll simply work my notice and walk out the door. Only the need to pay rent, and not fuck my career completely, is stopping me writing that letter now.
# posted by SaneScientist @ 3:10 am
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