Time flies when you’re having fun

I’ve mentioned before that my husband lives in another state hundreds of miles away.  We try to see each other for several days about once a month.  It looks like August will not be one of those months because he’s going to be here in late July for various reasons, then needs to travel for work in August, plus my sister is having a baby in August and therefore I am going to want to visit her then.  So, we are talking about the second weekend in Sept.  And this time, I’m going there.

And as we were making these plans, I was thinking about my labwork and I was thinking something like, “Well, I’d like to have a Committee meeting [aside, in my program, we have thesis committee meetings once a year or more and we have to have one before we can set a defense date] in late August or early Sept., so visiting hubby right then might not be the best idea because I’m either going to be stressed out because my meeting is soon, or I’m going to be stressed out because I’m finally writing my thesis.”  And then I realized that, unless some kind of miracle occurs between now and then, there is no way I’m going to have a Committee meeting in late August or early Sept. because it is already the second week in July.

Where the hell does the time go????

Okay, here’s what I have to do before I can have my Committee meeting (skip this part if you’re not into biology:

  1. Finish that cloning that is giving me hell (a week???).
  2. Clone something else in that construct five different times (about a week if all goes well, which it won’t).
  3. Finish a different cloning project (a few days, I hope) so I can make the yeast strain for the constructs in 2.
  4. Transform the construct from 3 into yeast.  (3 days for the yeast to grow up, one day to restreak, one day to make a patch [these steps are necessary for making sure I have a pure strain] and one or two days for screening the yeast to see if they are what I want.  Then mate it to make it diploid (3 days).
  5. Transform the constructs from 2 into the yeast strain from 4 (same amount of time as 3, or maybe a little more because the screening will be more complicated).
  6. Sporulate the yeast from 5 (2 days). Count spore products (1 day).
  7. Clean up and make strains from the spore products in 6 (3 days).
  8. Do microscopy experiment from the stuff in 7 (3 days–if I do them all at once, but since there’s a lot of them, I probably won’t be able to, so let’s say 6 days).
  9. THEN there’s this western blot stuff I have to do, and a protein purification I have to do which will probably take me a couple of weeks, BUT, I can’t start it until someone else in my lab has been able to purify this other protein (she’s gone through hell and back with this protein, and I’m glad she’s doing it, but it does suck that my work is dependent on someone else’s).
  10. Write a committee report (3 days) and give it to my committee one week before my meeting (7 days).

SO.  Total time is: minimum of 6 weeks.  Yes, I know my math is off, but I’m counting on being able to do some of this at the same time (like the stuff in 10 while I’m making the yeast strains).  If all goes well, I could have my committee meeting at the end of August.  BUT, seeing as how I’ve been around the block a few times, I know not all will go well.  In fact, some of it is going to suck (witness the fact that I am still working on a cloning project I started at the end of MAY; let that sink in for a few minutes).  So, if we double that time because that’s usually the sensible thing to do in labwork (calculate the amount of time you think it will take you and double it) we’re looking at 12 weeks which is THREE MORE MONTHS!!  Then, I have to take 2 months to write my thesis and then another two weeks to prepare and write the defense which means that–

Forget it.  It freaks me out to think this way.  Needless to say, I’m make the arrangements for the trip in Sept.  I don’t think it’s going to interfere with anything and considering the number of hours I put in at lab, I need to get away every now and then to keep myself sane.

Evil SOB

One thing that happens constantly in labs that do molecular cloning is transforming bacteria, that is, making bacteria take up foreign DNA.  The way to do this is to first mix some DNA into a small amount of (transformation) competent bacteria (usually E. coli–a non-infectious lab strain; transformation competent bacteria have been treated with some chemicals beforehand) and let them sit on ice for 20-30 minutes.  Then, you “heat shock” for a little less than a minute by putting them at 42 degrees Celsius (107.6 degrees Fahrenheit) then back on ice.  This essentially freaks out the bacteria and they start taking random stuff inside the cells, including (hopefully) your DNA.  Then, you want to incubate them in a really nice, rich medium for 30 minutes to an hour to let them recover from their ordeal.

Enter the SOB.

The media that we use is called SOC.  Now, we have someone in the lab who makes our media and pours plates for us.  But, she can only do very simple things (take this packet, dissolve it in water, autoclave it) and SOC involves mixing several things and adjusting the pH.  Which means we are on our own for the SOC.  On top of that, SOC contains a large amount of glucose (sugar) which will break down if you autoclave it, so, you have to make it without the glucose, autoclave it, then wait for it to cool, then add glucose.  Before you add the glucose, the solution is known as SOB.  Which always gives me a little giggle.  I’m childish that way.

Wow, that was a long explanation for such a short punchline.

Anyway, I decided the undergrad needed to learn how to make SOC because, well, he’s an undergrad and he needs theis kinds of drudgery learning experience.  Unfortunately, once we put the stuff in the autoclave, he had to leave, so I’m left to finish it off myself.  This is really not how I thought this would end. *sigh*

Cloning is sucking my will to live

Okay. Here’s the deal. The reason I started this here blog is because I needed a place to rant. I decided this after a particularly nasty debacle with mislabeled, poorly made vectors which I alluded to here. I started this blog about two days later, so that should give you some idea of the timeline I’m working with here.

Since then, I have been trying to clone an insert into a pUC19-based vector (which has been properly constructed–I checked). The thing I’m trying to do is put in a PCR fragment with a BamHI site at the 5′ end and a blunt, phosphorylated 3′ end. For various reasons, I can’t use any of the blunt cutter sites in the MCS, so I have been cutting with SphI and blunting using T4 DNA Polymerase.

It won’t work. I have tried and tried and tried. Why it doesn’t work is a complete and utter mystery to me. I suspect that the blunting reaction isn’t working as planned. I much prefer Klenow but that blunts different overhanging ends and T4 is supposed to be the one to use in this instance. I have done this kind of thing millions of times in the past, I don’t understand why this one is so damn difficult!

My apologies to the people who have no clue what I’m talking about. I just needed to vent my frustration about this stupid thing.