Stoopid Stoodents

Gobble, ST and myself were talking about this new undergrad project student who was working with us. The same one who took my set of Gilsons and put his name on it, even though he is only with us for two months max.

Anyway, this student has been really really really testing Gobble's patience. As in, he uses every reagent that she has, and then goes on to use everyone else's reagent (this has happened for antibodies, MW markers, films...).
As in, he plates out cells onto coverslips, and does immunofluorescence...without checking that the cells have actually stuck down. (Oops, there goes 200 pounds worth of antibodies down the drain!)
As in, he shuts down the hood and does not replace the hood cover. The hood cover might only be useful to keep cats and dogs out (and Drosophila or Daddy-long-legs would get in...they probably would even with the hood on...), but it's a nice touch to put that back on.

Apparently, when he started, he also lacked the ability to take down notes. A couple of people who had taught him noticed, suggested him to take down notes, and he still didn't get the message. According to Gobs, he is very good at calculations - can calculate molarities and stuff, real quick. But when it comes to the detail of the protocol (very important) he forgets. I mean, he forgets to put cells in the wells, for chrissakes.
And what's worse is that he doesn't admit to his mistakes. Whenever Gobs asks him "have you done X?" his first reaction is "yes". She has to ask him three times before he admits to forgetting it, or not doing it....just telling her the plain facts.
I can understand that part of it might come from apprehension to Gobs' reaction - he might be scared of it, he might be worrying etc., but as a project student you should be in the lab to learn. And make mistakes. And as a supervisor, Gobs' job is to point it out.

ST thinks that it's just students in general. When she showed Dolly some technique or other, she noticed the same thing with Dolly - no note taking. And whatever notes and protocols that ST gave her, the next time in the lab, Dolly would "forget" it on her desk.

ST thinks that it must be students nowadays - they just don't take notes anymore. They are given handouts in lectures, given protocols in labs, all reagents are ready for them. Too much mollycoddling. I think there is a lot of truth in that.


I don't know what is the best way to deal with this kind of stuff. I remember when I had that stellar project student to deal with, I knew she didn't have any experience before in the lab, so I told her that I would do the procedure three times with her - show her once, do it together the second time, then watch her do it the third time.
I also remember threatening another PhD student whom I had to teach, that she better take notes down because I am not going to show her twice. I've since mellowed a bit, and don't say that, but since the student still remembers me saying that to this day, it must have worked.

It probably is better to lay down the rules before starting, like "I am not going to repeat myself so you better write EVERYTHING down". And make sure the student understands who is the supervisor here - the student is only there on your terms, not his.
I only lay down the rules first because I don't want any misunderstandings that might stem from the fact that I am a foreigner. So by being honest, I cut down on any misunderstanding that might stem from miscommunication (through difference of culture, and not language, for me).

It's all about covering your own ass. Really.

Last few posts

...before going on holiday, from me.

It's never a good thing to read other people's blogs when you are supposed to be packing, but when you don't have a lot to pack....well. Anyway, there is an interesting post over at DM regarding authorship on a paper. I think I may have blogged about this before, but I might as well do it again since I can't find it.

I have a first author paper, in which I had a minor disagreement with the PI regarding author rank. It was mostly my work (actually, in the initial stages it was all my stuff). I left, and it was left to the CM to finish off the work for the paper, which, of course, you have more after submission because of the reviewer comments.

Anyway, CM was already listed as the second author, since she made the knockout mouse, where I got the cells from. Now, CM is slightly more senior than me, and already had a first author paper from another knockout mouse she made (and her publication list was longer).
When it came time for resubmission, the PI said that she thought CM should be the first author. To which I totally refused, stating that (1) the initial work was all mine, (2) CM has a first author paper already and can have this paper as "joint first author" paper (I agreed to that, at least), (3) CM has more publications and I needed that first author paper.
I did threaten my ex-PI that I thought it was unfair to change the order, and that I would just take the manuscript and publish it elsewhere (at a much lower impact factor journal). Thankfully, it didn't come to that.

What helped was that CM agreed with me, that I should be the first author on that paper. And I think she did mention that point to the PI. Otherwise, I don't know what might have happened. As it is, the paper got published, and my name is first.
I think part of it was that the PI came from a lab, where the producer of the knockout mouse always was first author on the first paper published using that model. I think, as a postdoc, the PI's name got bumped to second because she was using a knockout mouse made by someone else.


Another thing - I commented about it in DM's post.
I have a middle author paper, where I did a western blot for the project. I wasn't actively involved in the project (bar asking the people involved, "how's it going?"), but at the time, I was doing westerns all the time. The PI must have liked my westerns - the lack of manky spots, the crisp bright bands - because he asked me if I could help them by doing a couple of westerns.

I think he did make it clear that my name would be on the paper - if not him, then the lead postdoc working on the project did say so - but I wouldn't have minded doing a couple of extra gels anyway, since western blots aren't that hard to do. And it made sense, since I was doing it all the time, and had experience with the antibodies.

So there you go. One first author, sort of wrangled, and one middle-of-the-pack, which I am more than happy with, for a price of a pretty figure.

Part 2

From the previous posting.

Anyway, the point is - you would not believe the patheticness of our IT services department. So here I was, needing to update my SPSS from version 11, which I had installed when I first started working here. Okay. What I needed was the new software - I knew we had a institute licence, but I just needed to upgrade my software.

So I did what most people would do. Contacted the IT services.
And they took a good few days to talk to me, to tell me about how they can upload it onto my hard disk directly if they had my IP address, and how if that didn't work they would send someone down.

That took a weekend. And still no new SPSS on my harddisk.
I sent them my IP address. No news at all.

This coincided with a time when the IT services were trying out this new software distribution system, which was just about coming online. So I thought I would have a go.

It was 90 minutes of my life wasted, really, but compared to the two weeks I probably would have to wait for the damn tech to come up to my office to install it, it was 90 minutes well spent.
At least my manuscript has gone now, with new little asterisks on the bar graphs to emphasize the point.

But the thing that pissed me off was, (1) it was quicker for me to sort shit out on my PC than wait for the damn IT people to get their shit together, (2) the instructions pdf files for "how to get your downloaded software installed" was so shit that it took me a good 15 minutes to figure what the fuck they were talking about, (and I have a PhD so I know how to think), and (3) they took a hell of a long time to get back to me to tell me all that. Like, they were trying to get in touch a week after I sorted the shit out.

Ugh.

I also hear that they want to decrease people's accessibility to the PC - disallowing administrator access to the new PCs. As far as I know, they did that for technicians and students - do they do that for research staff too? Because that would suck.
I can understand the need to limit access, so students (ahem) don't put filesharing software on the institute owned PC. But you know when you are doing research, sometimes you want to download and install a small software that you would probably be only using once or twice, just to scratch that proverbial itch. Well, I do. And that would just make it hellish for me if that freedom is taken away from me.

All by myself...

Being from the branch of science where statistics is known as something social scientists do and hence was not taught in undergrad courses (i.e. chemistry), I had for a long time avoided dealing with statistics. I normally give my data to someone else (i.e. bosses) to do the stats.

I hate stats.

All those tests confuse me. Why can't we have one test to do so that we can say that "this data is significantly different from that data"?
It was all so simple in chemistry. If you tried to make something, it was normally there. If it wasn't there, then it was something else. Or the starting materials. But it was still there.
In solution kinetics which I excelled at, there were basic rules that the molecules followed. And I say rules in plural, because sometimes your solution follows a few rules, not just one.

Try saying that to cells. No, they are not happy to follow rules. They think, um, ah, what should I do?
Aw, maybe I'll die. No, maybe I won't.
Aw, maybe I'll be transfected. No, don't like the plasmid or the transfecting reagent, I'll die instead.
Aw, maybe I'll produce all these proteins. Naw, I feel sick and the scientist left me out in the open for too long and the media is turning pink and......ughmph (or whatever noise cells make when they die).

Anyway, I had to do stats. And now I know how to do them in SPSS. Well, I know how to do one test for my one particular luc assay experiment. And that means I can put asterisks on my graphs.
I think that was my one big achievement last week.

Scientific stardom

Over at FSP, there was a post about rockstars and scientists. And it reminded me about the Nobel Laureate I met.

A group of us from work went out to a bar after work, just for a farewell drink with one of our Ph.D. students. He's not leaving leaving, because he is still working in the same institute, but in a different lab.
Anyway, one young PI who joined us recognized a Nobel Laureate ordering food at the bar. He claimed that that scientist type guy standing at the bar was none other than John Sulston, recipient of the Physiology or Medicine prize in 2002.
Of course we didn't believe him, but the buzz was there. Is it him? Should we ask him? Should we dare to go up and ask him if he is THE John Sulston? (As you can guess, we don't know our Nobel Laureates that well...)

After much trepidation (and typing feverishly into our mobiles to find his photo on the internet) on our part, the Farmer went up to him to ask. And it was him! He turned around, looked at us, and waved!! We got him to come have a chat, and had a photo opportunity, which is where this photo comes from.
He was very nice, BTW, very friendly and down-to-earth (considering he was conversing with a group of young-ish, gaping scientists who were so awe struck that they couldn't form a sentence). The kind of person you would enjoy talking to in a pub after a conference session, for example.

We were wondering then if he ever had this - you know, some random person coming up to ask "Are you THE Nobel Laureate???!!!" in a bar or a pub. I guess that if you are a rock star, that would be a common occurance, but a scientist? I mean, that must make your day. Unless you are a Nobel Laureate, and you want to keep a low profile when ordering some food in a bar. He had a bowl of fat chips with whatever he was eating, by the way.

BTW, if there is one Nobel Laureate I would recognize, it would probably be Sir Paul Nurse. Only because I have actually seen him give a talk and all, so have a good idea of his physical appearance.

What's frugal?

Following on from the previous post - if there are things I am wasteful of, at the moment in my project, would be tips and eppendorfs. I think I went through about 3 boxes of tips today, all in tissue culture. When doing luciferase assays, I can go through another 3 boxes of yellow tips, just for aliquotting out the samples.

If, during a serial dilution, I get distracted and lose track - I don't sit and think about where I might have made a mistake (if it is that hard to tell by eye), I throw it away and start from scratch. Far better to be sure than to be guessing.

I do a lot of compound dilutions, and as I do these assays in 96-well plates, the volumes involved are so small that I actually do make dilutions in 96-well plates instead of 1.5ml (or even 0.5ml) eppendorfs. And when you are trying to pipette 10ul in all the wells of 10 x 96-well plates (from dilutions you make in 96-well plates), the one thing you don't want is a mistake.

The way to do that for me, then, is to make everything orderly and prepare everything beforehand. Preparation goes a long way to minimise confusion and mistakes. Even if a person might ask me a question when I am in the middle of something, I normally am able to answer them cordially without getting confused. I got asked a question like 5 times today, all in the middle of adding something in a 96-well plate. And all the wells look so much alike....

So it is simple things like keeping the hood clear of clutter; collecting everything you need, to be where you need them (and that means racks, gilsons, pipettes...); deciding and sticking to a certain format in plates; liberal use of markers and plate lids (my lids are normally crammed full of writing, and it doesn't help that I have a relatively large handwriting); knowing one's limits (and the cells' - if your cell type don't like staying out in the cold, unbuffered atmosphere of the hood, then don't leave it out there for long).
Oh, and taking a 10 minute break every hour or so.

And if I do get confused, I start from the last possible certain point (a bit like a save point, I guess). I do make mistakes, but at least I know where I made them. To make sure that I know where I made them, is I guess, where experience comes into play.


If there is one thing I am greatful for, is the electronic pipette. I normally do most things with a normal gilson - diluting compounds and stuff - because I am quicker than the motor. But when you are aliquotting out cells, or pipetting 10ul per well, in triplicate, in 96-well plates, make that 10 x 96-well plates....electronic pipettes are the way to go.

Just Do It

Sounding a bit like a familiar brand here.

I thought about this, since I was talking with Mac today about how to go about stuff - he's testing some stably transfected cell-lines to see if it works as well as my transiently transfected cells. There are a few compounds to test, and he was asking me what he should do.

Basically, he doesn't know whether the stable cell-lines express the reporter well enough or not. I heard that a stimulus will only give a few-fold increase, and that isn't what I want (I want a bigger one!). He's set up an experiment already, with the control stimulus, which he has yet to read the luc reading for. But he has several plates all ready to be tested.
The question was - should he use the plates and test some of the compounds, even if he hasn't got the result for the crucial one (i.e. before he knows the effectiveness of the cell-line as a model), or should he wait until he gets the result?

My answer is - just go ahead and add them. The experimental set up is such that after stimulation, the plates can be frozen (without lysis). So it is a situation where you don't lose much (bar some compounds, and your time). If the luc assay for the control stimulus doesn't give the required reading, then trash the other plates. If the luc assay works, and he (or I) am happy with the result, then he has saved one day (and some plates of cells) and got a result.

This kind of thinking, where you just try it anyway, even if you know it might be a waste of time, only came to me with age and time. I am not by nature someone who likes to take a gamble - I like everything to be working and orderly.
But if there was one thing the Big Boss has taught me, was to try and just get the result as quickly as possible, even though you might be wasting (a little) reagents.

I guess this kind of thing comes with experience - research as well as life is not a linear experience, and sometimes you have to gamble; sometimes you have to jump the gun. (Especially when you have a finance officer who doesn't order your stuff regularly...)


I don't paritcularly like wasting reagents - even plasticware. Although when culturing cells, I always try and have a flask extra "just in case", I normally don't have a lot of flasks to waste. Sometimes, like when there is a mycoplasma outbreak, you just have to waste it - there is no point being frugal at times like that - but the differentiation between frugality and free spending, I guess, is something you learn with experience.

Correct pronounciation

Whilst talking with ST and Mac, I realized that I pronounce restriction enzyme names differently from Mac.

ApaI = "ay-pah one" (me) and "ah-pah one" (Mac)
HindIII = "hindee three" (rhyming with Indy - me) and "high-nd three" (rhyming with behind - Mac)

I can't think of any others at this moment, but there are sure to be others...

Three heads good

Why is it that people seem to have this urge to re-name plasmids? For fuck sake, keep the name the same! If the maker of the plasmid deemed okay to name the plasmid a boring "pXYZ 13", use that fucking name!! If the maker of the plasmid deemed okay to name the plasmid "pABCD-EFG-HIJ" and you find that with your dirty handwriting, you can't fit it onto the eppendorf, practice writing smaller!!

I am sure that this re-naming of plasmids is the reason for our plasmid mayhem.

Anyway, as the title of this post states, today I had a meeting with Mac and ST, regarding our problematic plasmid. We are supposed to know what this plasmid is. It certainly does what it is supposed to do - it is a reporter construct with a Luc gene in it, and when I put it in cells and induce it, I get a reading for the Luc assay.
But the restriction digest is troublesome.

HindIII cuts once. I did that, and it (seemed to) cut.
Of course I didn't run the un-cut plasmid (because I never do these things, often it takes me three times to realise that that is a good idea).

ST tried cutting with HindIII and XhoI.
It cut once. It should cut out the insert. The whole reason why ST chose to cut using those two enzymes was that there was a note on the plasmid information page (in the oh-so-well kept plasmid files....not!) that read "HindIII and SmaI should cut out the insert" and that the SmaI site was inside the XhoI site (i.e. closer to the HindIII site on the multiple cloning site). So in theory, it should cut out the insert.
If it has an insert. But it has an insert, because I can induce it.

Mac tried cutting with that funny enzyme, ApaI. It didn't linearize.
It should. The ApaI site is not in the MCS, it is somewhere along the plasmid between the resistance gene and the Luc gene. It's there, and it should cut. But it didn't.

So people, even Sherlock Holmes will find this troublesome. Actually, he would, since he didn't have restriction enzymes.

Anyway, as we have a more pressing need to know what this plasmid X really is, we are going to try again and cut. And this time, send the darned thing to sequencing.
Sequencing should be easy, because this plasmid has been published on a paper detailing it's origin (actually Big Boss made it, and he made it by cutting the promoter region from one plasmid and stuck it in the Luc plasmid), and the sequences can be obtained from PubMed. So if we design a primer going upstream from the start of the Luc gene, we should cover whatever is inside that MCS...

You might be wondering why I didn't do that in the first place.
You see, I sort of took another person's word for this plasmid because I was really pressed to deliver results by the Big Boss (so indirectly it is Big Boss' fault). And anyway, Big Boss should really tell the other person to keep better records of the plasmids - that person has been working with BB the longest, and BB somehow implicitly trusts him. It seems like BB can't get rid of him now because he holds all the crucial information as to what plasmids are what and where they are in BB's vast empire.


The upside of this sorry circular (har-har!) tale is that Mac, ST and I had a good laugh about how this kind of thing would be good either as a final-year undergrad or a short-term M.Sc. project, or an exam question for a Mol Bio degree.
"Here is a mystery plasmid, with this bare backbone of information. Find out what it is"

It's plain plasmid archaeology.

Fearsome reputation

It seems that there are thiefs about in the lab. Okay, let me rephrase that to "colleagues who lack responsibility". Fuckers.
Anyway, this person apparently used the last of the luciferase assay reagent from Gobble's stock. And instead of throwing away the empty box, or telling Gobble about it (after ordering the replacement, of course), this person just left the empty box in the freezer shelf.
Gobble was not happy.

Now, I happened to walk past, and asked the topic of conversation of which Gobble was engaging Elle, and that was the reason. At that point, I was about to offer my meagre supplies (which I am not particularly happy to share, since my finances are scrutinized for this current project), to which Elle replied that she would be alright for the time being.

I was pondering about this, and thought that I must have a reputation since no one actually "borrows" anything from me without asking. I was discussing this with ST, and we both came to the conclusion that I have a fearsome reputation - no one pisses me off (in that way), and if they do, they will have to deal with the WRATH of Lou.

Which is, ST and I both agreed, was a good thing.

See, I am happy to share reagents, and be helpful to anyone requiring assistance. I am generally nice to people (okay, cordial maybe), and won't (most of that time) shoo them off when busy. I have long learned that when one is trying to pipette 10 different reagents to a 96-well plate and someone talks to you, the best thing to do is ignore them for 10 seconds, then tell them you are busy (like they haven't figured that one out yet).

I also do hold grudges for a long time.
And my natural facial expression must be one of irritation, judging from how I have been treated when I kept my facial expression to neutral (or what I thought was neutral). I must look pissed off when I am just....standing there or something.

I'm also wondering if my general sarcasm and pessimism to human nature, as well as my wonderful intelligence, makes me a force to be reckoned with.

Oh well, in any case, think I'd like to keep this reputation.

WoT part 2.

I'm still irked from yesterday's seminars.
So I am thinking about the best way to put down a (first-year) Ph.D. student in seminars attended by PIs (including the student's supervisor). How do you go about that?

I guess the best way is to have an air of mild concern and irritation. Say something in a neutral tone, not friendly, but not aggressive either. Show concern about how the student may have had little time to prepare for the talk (which can be a back-handed way of telling her that "even if you prepared it well, it was a shit talk").
What would I have said yesterday?

To speaker 1, as I know her from Journal club, I guess I could comment on her talk before one such occasion. Shame that we aren't having anymore JC's for the summer. Anyway, my major concern really was that in her western blot, the protein was showing up about 20kDa above the expected size - which could mean that it is unspecific (species was different to the one Ab was raised in, so a possibility - and there were no controls. Actually, I don't know whether she did the same species control, which would have been the most obvious and easiest control, since everyone uses a human cell-line), or a post-translational modification (less likely).

To speaker 2....now that it a challenge.
"Okay, first of all, do you know how to use a graphics package like Adobe Photoshop? Because after you scan in your blots, you can rotate the imported scan to make the bands horizontal? And you can also change the blots from a colour figure to a grayscale one, which will make it easier for us, the audience, to see whether the band is there or not. And also, in Photoshop you can change the contrast and intensity to make is easier for us to see the difference in the bands.
You see, saying "trust me the band is there" - I can accept it from an established researcher who has shown other good quality blots in the talk. But I find it hard to accept it from you. I don't know you, or the quality of your daily experiments, and you might as well have brought the film in to show us to convince us.
I understand that you may not have known the audience expected for this talk, which is actually your responsibility to find out and to aim your talk to. And I can understand that you may not have had the time to fully prepare for the talk.
I think your talk was good, with a great introduction. But it is a real shame about the results, because you could have done a little more to convince the audience."


Granted, it's a pretty hard thing to say to a 1st year.
But I don't get paid to sit in a seminar that is shoddy, when I could be sitting in front of my plate reader reading luciferase assays.
Of course, I probably would never say that in a seminar full of PIs, since I am only a postdoc *sniggers*.

WoT

There is nothing more exasperating than going to a talk given by first-year Ph.D. students. They know plenty about the topic but they lack the finesse in the art of giving a talk.

I had to attend two of them today.

And what a Waste of Time it was. I really wanted to point out the finer points to both of them, but since I didn't want to appear too aggressive, or off topic (I wasn't going to comment on the science, although it wasn't totally atrocious), or make a fool of myself to the numerous PIs in attendance (who have by now learned to observe in an encouraging manner), so I kept quiet.
Thank God I have a blog. I can rant now.

To speaker 1:
a) Don't use serif fonts when presenting in powerpoint.
b) Don't talk fast. Fucking take a breath. (I was about to put my hand up and ask her to speak slowly, it was actually annoying me)
c) Do a proper introduction to your topic - especially since you are an internal speaker, you should have been able to guess at the audience type (varied). It helps to explain the terms you are using, you know? And go through your model properly.
d) Don't show a western just because that is the only data you have. A western whereby you are testing primary antibody concentrations to use, is not proper data. It's more like...preliminary data, in the mildest sense.
e) Explain your slide and figures on your slide. Take time to. We can't see that tiny dot with the name of the gene next to it, on that two-axes microarray results, when we're sitting at the back of the room. You might be able to, being 2 feet away from the screen.

To speaker 2:
a) Don't apologise about the quality of your western. It sucks. We can tell. Your technique at the moment is, to put it mildly, SHIT.
b) When you scan in a blot and the bands are at an angle, do you know what you can do? Use Photoshop to rotate the blot. Yes, rotate, so that the bands have a semblance to the horizontal. Not doing that shows that you are not versed in computers and graphic software, and that you aren't thinking about the audience.
c) Again, spend some time introducing your topic to the audience. You don't have any decent data to show, and we know it. So don't bother showing us every little experiment you did.

I understand that they are first year Ph.D. students. But I have long passed that stage in my postdoctoral life when I can be optimistic and nurturing to these students (who aren't in my group, BTW). It just pisses me off that I have to sit through a talk given by a student who obviously didn't ask anyone for advice about talks.
It is so simple - just explain fully your project. Everyone knows you don't have decent data. Everyone knows you are a first year. Don't do cocky. Don't be dismissive about your audience.

In the end, I just thought that they are so arrogant - as in, they don't think about the audience at all. That pisses me off most of all. You don't have to sell your project at this point in your career - just present it well. If anything, the audience would have come out of it learning something new, not about your project, but another research area that they weren't aware of.

Journal club woes

I am reading a paper which is like an example of "how not to present data".

As in, don't just put bar graphs for 5 figures. As in, if you are showing the same data as in the supplementary figures, just show the fucking data without the spin. As in, if you have the western blot, don't do densitometry...or at least make the blot the centrepiece of attention, not the densitometry values. As in, if you are showing positive fold and negative fold (i.e. 4x or 1/4th) use a log scale, especially if you are talking about "fold" in the text.

You know when you just start picking on the actual presentation or grammar of the paper? And it's not like I was trying to - I mean, all this epigenetics (I hate that word with a passion) is not something I am familiar with, so I was casually reading through the paper. I tried.
But by Figure 3, I was finding holes. Not even with the paper, with the presentation of data in the figures.

Taking into account my intellectual standards when I read a paper, this current paper is pretttty baaaaad.

Ow...

Now that I have my electronic pipettes, pipetting is so fun. I can sit there and just pipette away until the day is over. But (yes, there is always a but) I get a very sore elbow.

Not from bad posture.
Not from extensive use of my forearm.
But because of that darned grill at the front of the tissue culture hood.

I need an elbow patch. An elbow cushion. Maybe an elbow-attached version of this. Maybe I should get one of those pads for inline skaters. Or this. I mean, I AM dealing with an "extreme tactical situation" here. Surely.

I am invincible!!!

Spent the whole afternoon in the t.c. room today. Thank God it was raining outside and cool, because I would've DIED if it wasn't.
And thank God for electronic multichannels. Those thirty 96-well plates I did today would've broken me (especially since I started doing them three hours into my t.c. room stay - yes, after doing twenty seven 24-well plates, by hand, by normal gilson). It probably took me around an hour, slow pipetting, to do them all.

My thumb is a bit numb right now.

Man, I can't wait until I get my Finnpipette Novus pipettes.
I love their trigger mechanism. It took me a while to get used to it, but it is so unbelievably great once you do. And when you have to use your thumb for pipetting everything else, the forefinger trigger is the way to go.

How not to argue with reviewers

So my manuscript came back again, with comments from 2 reviewers, which are relatively easy to work with. So I think we will be sending it there. Hell, it's easier to address these reviewers comments than the previous ones from other journals - at least we won't have to work another 5 years and write another grant to publish it.

Anyway, it got me thinking. Reviewers mostly asked for the same stuff. But what if I wasn't feeling normal when answering them?

Q: Why did you use this particular cell-line?
A: Because someone in the lab was already culturing it. Having endogenous protein X has nothing to do with the model. Nada. Zilch. Duh!!!

Q: Is there a statistical difference between the effect of compound X and the control?
A: My stats package...sorry, Excel, didn't have the Whitney-Freeman-JoeBloggs stats test. Yeah, okay then, we'll come up with something for that bloody p-value.

Q: The westerns are not of good quality.
A: Well, why don't you try running a western with tissue samples.

I was thinking of more stuff whilst working on the manuscript at work, but I've forgotten them now...

Lab porn

For me, it's companies' catalogues. I love shopping. And it's better when it's not your money, and you can buy hundreds of pounds of stuff.

5 x 1ml vials of transfecting reagent, which costs 1500 pounds? Got it.
Electronic pipettes? Got it.

Oh, it's so fun.

I'm feeling quite happy in my job actually. I don't know whether it's the equipment money factor or the actual project (quite simple)...

Not an ad...

Emptying bins around my hood of full clinical waste bags and full autoclave bags, and putting new bags in - 5 minutes.

Making up new stock solution of Virkon, and fill up my empty wash bottle with it - 2 minutes.

Gathering all the pens and gilsons and pipettes and tips and plates (from various locations of the vast t.c. suite) required for extensive t.c. session - 5 minutes.

Going to stores to buy the 10cm dishes I need, which has run out of the t.c. suite - 10 minutes.

Switching on hood and wiping down all surfaces with Virkon and ethanol - 7 minutes.


No infected samples - priceless.


Our tissue culture room is a tip. I mean, seriously. I heard that Gobble had some mycoplasma problems, and I am not happy with that - we share hoods and incubators. Our hoods don't have UV light. Ho hum.

Slogging it

If it wasn't for good ol' Canter this morning, I would've quit.

I was making an agarose gel to run my restriction digests this morning, and I couldn't find the agarose I used to use before. I couldn't find the normal agarose on the communal shelf. So I took the nearest agarose, and was at the balance to measure it, when Canter called my name.
He then told me that they've been having problems with the agarose, and to use a particular one (which was decanted into an ordinary bottle, so I didn't trust it).

You know, he didn't have to say anything - I guess he just noticed me taking that other bottle. I think because I am so used to people being nasty, consciously or subconsciously, I was sooooo greatful for his little help. Seriously, if he hadn't stopped me using that dodgy agarose, I would have just quit for the day. Not that he knew that.

So that was a nice start to the day. I got to run my digests - most of them were correct, which was good. The two colonies that I maxi-prepped blind (i.e. before running out the digest) was as I expected, so that was good - I thought I might have to chuck away a maxi-prep, but my anticipation was correct.

Tomorrow is another day, and as I think of ways to tell my boss "I haven't done the stuff this week because I couldn't get hold of reagents", at least at the start of next week I should have everything ready to go. Including all my suspect plasmids, maxi-prepped.

The Little things

It was the little things that made me want to stay with the same boss, on this new project. And it's the little things that is making me think about moving to another job. One week back to the lab, and I am already thinking it was a waste of time.
It's not the boss, or the project. It's the little things in the lab that is making me think.

Let's see.
First of all, my transfecting reagent failed to arrive. If I don't transfect today, it means that I don't get to do a reporter assay until Saturday. And I am not coming in on Saturdays.
I just wish that the ordering people would just tell us when they are going on holiday, and what their contingency plan is. I don't want to have to order something, thinking it will be ordered, then finding out two days later that the person who normally does it, is on holiday. That is just not acceptable - it shouldn't be my problem. Actually it isn't, but affects me.

I was asked (nicely) by GorillaGrrrl that the tryptone and yeast extract on the communal shelf was hers, and that she was surprised that people were using it. I told her to label it, and put it on her bench.
She also mentioned that when she moved in to the current lab, there were only 3 PIs, and that shelf was hers. I told her, "oh", insteady of "that was 4 years ago, things have changed, move on, and anyway, where were you after your maternity leave for six months? Oh, and if you were so worried about other people using it, why didn't you tell your student - the last non-GorillaGrrrl member of her group, who left a year ago - to keep it on her bench?"

She also pointed out that one of the Gilsons on Doc's bench was hers, and she took it back. She only asked moi, because she thought Doc's bench was mine. Well, fuck you too dumbass - I hadn't been standing in front of Doc's bench to do my morning's miniprep work, have I?
Anyway, someone probably inadvertently put it there, since I hadn't seen that Gilson there before (like, last week), so instead of just taking it, you have to make a point.
As you can probably guess, I am not fond of the GorillaGrrrrl. She "makes a mountain out of a molehill" according to our own ST. She just says certain things to make a point, and a big deal. I'm glad she ain't my boss. But with her insecurity, she probably won't employ an experienced, intelligent postdoc like yours truly. Ahem.

Then I found out that we were lacking a few agarose gel trays. I swear that when my contract finished last year, there were at least 2 large agarose trays still functional in the lab. I can't find them anywhere now, and I asked others but they seem to recall that it may have been broken.
Gees, if you break something that is communal, shouldn't you replace them from your grant? I can just imagine who it was (or more correctly, which PI's group) did that.
Anyway, I just couldn't be bothered anymore. I did my mini-preps, all 40 samples. I did my single restriction digest. But I couldn't run my samples today to double check whether the plasmids were what people said they were.


Oh, and good thing that I check plasmids. I am beginning to wonder why people keep insisting on calling plasmids the same. Lets say we have the ABCD reporter gene plasmid. Say ABCD is a well studied promoter region. Of course, people are going to make all kinds of different deletion to see which part does what. And the thing is, now, I have in my hands 4 different ABCD-Luc reporter plasmid, all with the same name.
I know one was made by someone in our lab. One was given to us by Dr.L. But there is another one, again made by Dr.L, but as far as I know, the plasmids are different by 1kb (so my guess is that the latter is a deletant of the former). Then there is another one, which seem to be a separate deletant.......*exasperated sigh*
Why are people so boring when naming plasmids? Can't they call it something snappy? Can't we just call them ABCD-Luc Alex, ~Bob, ~Clyde and ~Dave? Like the TV channel. Or Angie, Barbara, Connie and Debbie. The name can be the person who made it, or the person who sent it. Or the place. How about pSB for Santa Barbara. Or pTex for Texas. Though that makes Utah a bit problematic. I know, just don't ask for a plasmid from someone working in Utah.

Well, then there are the things I mentioned to Santa yesterday.
Today, at 4pm, I felt pissed off because I had to keep an eye on the sole bug shaker so that the two empty clamps would be free for me to put a couple of big flasks (for a maxi prep) in.

I dunno, maybe I'll just drown my irritation in some beer.