Sometimes I think the universe is trying to determine just how far I can be pushed before I completely lose my mind. I can tell you right now, it’s not that far. In general, my fuse is pretty damn short. Patience is not a virtue I possess and I do not fear or avoid confrontation. However, I really do try to keep myself under control, especially at work. If you compare myself of today with myself of 10 years ago, you would find significant improvements in this arena, but if you compared me to myself of just one year ago, you would probably find little improvement.
Then there are weeks like this, where you might find it difficult to see any progress no matter which version of myself you compared me with. It feels like 10 million little things are going wrong. No, none of them are worth losing my mind over, but as they add up, I find myself dangerously close to the breaking point.
As an example, let me describe a 1-hour portion of my day yesterday:
- Our lab possesses multiple thermocyclers and right now, we are down to one. I think fixing one of them would be nice, you know, just in case that last one decides to die. Yesterday, the last thermocycler died. Fortunately, it was some sort of issue with a fuse and was repaired quickly, but considering I cannot do what I need to do without PCR, I was pretty damn irritated.
- I send DNA for sequencing. I get results using one primer, but no results using the other primer. I use the sequencing facility's primers. If it were my DNA, then neither primer should have yielded a result. Therefore, I think it is their primer and that they should re-run the samples for free. They are not under that impression.
- I open the -80 C and a mountain of tubes fall all over the floor. Someone decided that balancing an open box precariously near the front of the freezer was a dandy idea. I want to leave their stupid shit all over the floor, but I pick them up and shove them back into the freezer.
- After picking up all the tubes and replacing them in the freezer, I try an locate the strain I need, only to find that whoever got into the stocks previously completely rearranged all the boxes and since they are covered in frost, I have to remove them one by one, open them and pull out a vial to determine which box I am in. No, I don’t fix the order, because I am annoyed as shit.
This kind of irritating crap has happened every day this week. I am so ready for the weekend. I need to rest and reboot. I really hope next week is significantly better.
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6 comments:
What primer set are you using? M13F and M13R? I find routinely that our M13F sequences work great, and the M13R reads suck.
Interesting. Yes, I am using M13F and M13R. In this case, the M13R worked, but the M13F did not, making me think even more that the sequencing facility should rerun my stuff.
I'm going to keep my eye out for that in the future though.
Another thing to keep in mind is that secondary structures in your plasmid may come up blocking the one end but not the other. is your M13F close to G or C strings? You are right the seq facility needs to rerun your samples maybe modifying the cycling parameters for the M13F reaction.
Are you by any chance using SeqWright? Their M13F fails most of the time. My conversation with them:
Anonym: What happened?
SeqW: Bad DNA!
Anonym: But M13R worked! Do you think that my F DNA is bad?
SeqW: Yes!
Anonym: Well. Thank you!
At that point I changed to a different sequencing facility!
Good luck!
dimitris - Good point. It could be secondary structure and sadly, it didn't even cross my mind. However, all the constructs I sent have identical 5' and 3' ends, and the primer worked just fine for half of them.
Anon - Man, that blows. I just don't get how this happens. I've sequenced with M13f and M13r forever and never had these types of problems. At my last place I could send out total crap and get back beautiful, long reads.
Update: I did get my sequences rerun for free. Even better, I've found a new company to try. They will pick up my samples and have a faster turn-around. I'm keeping my fingers crossed.
Although I'm sorry for your misfortune and frustration, I'm glad that I can read a blog post and think, "I've been there, and it sucks." I just might feel better about this happening the NEXT time... because you know there will be a next time. :)
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