100th oligo!

I ordered my 100th oligo since starting the new postdoc today. They've been used for placing GFP on my genes of interest, making recombinational knockouts, checking things are where they are supposed to and hopefully confirming things aren't where they are supposed to.

Can't say they've all been useful but some have served their purpose. Let's see how long it takes for the second century.

This week in the lab (part 2)

So I left you all on a bit of a downer last time. Trust me, it was worse for me and I had to wait 3 days for some positive news.

On Monday I sent the gel with SILAC samples on for mass spec. analysis. SILAC sounds terrifying but it's really very straight forward in principal. The key thing is that arginine and lysine can be produced as heavy isotopes - the amino acid is the same it's just that they are made from heavy stable isotopes eg deuterium, 13C and 14N. If you grow your test cells in heavy media and control cells in light media then you have a way of telling them apart (with mass spectrometry). Here's a diagram that I hope illustrates the experiment.

Simples?

I was really lucky in that I sent my sample when there was no backlog of samples and got my results back on Friday morning, rather than in 8 weeks time.
I (or more accurately in conjunction with colleagues more familiar with this interpreteting) need to pick through the results tomorrow. An exciting hit is for a protein connected with a human neurodegenetative disease (ND)
Now, it's well known that you're only ever one experiment away from destroying your project but this is exciting as it actually gives me something to work with and that's exactly what I needed.

I think it's called "hope"?

The fact there may be a potential link with a human disease is a generous dollop of icing on the potential cake.

The other potentially good news is that the only mutant I have, the viable one, is the mutant for this gene. It could well be that I haven't even spotted any movement problems in the mutants and I certainly haven't been looking at their brains. Now I have a reason to look closer. It might also have no phenotype in isolation.
The first priority is to confirm the interaction which I can do in several ways, such as;

1) Confirm I can pull ND protein down with "MyProtein". This only confirms a physical interaction though.

2) Confirm a genetic interaction. There are models of the ND in flies and I can use this model to see if "mygene" mutant modifies it. This would be even better as it would confirm there's a physiological interaction and whether it is a positive or negative regulator of the ND.

If test number two works then things are looking up. If not, I may be back to square one or looking at some of the other hits from the interactome. Let's see what the next experiments bring...

EDIT: Censored on the off-chance it's giving too much away. Damn paranoia about being scooped, but you never know. It costs people their jobs so I shall remain secretive until it turns out to be a waste of time, I publish it, or someone scoops me anyhow.

This week in the lab (part 1)

This week managed to capture the highs and lows of research.

On Tuesday my PCR suggested that I had an ideal mutant for "Project4". Basically the P-element gives the fly red eyes and when the P-element leaves the fly has white eyes. Sometimes when the P-element leaves it takes neighbouring DNA with it and can create mutants. The process is random and has to be muscularly confirmed. I began by screening the lethal candidates, as they are most likely to have a mutation - what with them suddenly being dead. One of the candidates looked like this;

All well and good as P-elements are only supposed to excise in one direction meaning "not that way" gene should be intact. However, I decided I should check this, just in case, and because i also wanted a stretch of DNA that covered the mutation for sequencing. This is what happened;

What the hell? So much for that theory of it only deleting in one direction. This is bad news as I have no idea whether the lethality is due to a mutation in "project 4" or "not that way!!!" gene. I'd have to put "not that way!!!" back into this mutant to show the effect was due to loss of "project 4" - if that is even the case. "not that way" gene is a polycomb related gene so it's easy to imagine it could be essential.

This pissed me off quite a bit as it's the second really iffy P-element excision I've had in this lab. The other mutant I've made for "project 2" is what I like to call the "quantum mutant" as it can only be observed indirectly. Basically the P-element is still there when tested indirectly but isn't there when I test it directly. "Project 2" and "other way please" gene are intact but the line is now lethal. The original P-element line is viable...

At this stage I wanted to knock all my fly stocks off the table in a "Bill Adama, wrecking his model ship/painting" rage. The only mutant I've made that makes any sense is viable  and it's hard to drum up excitement for a gene that doesn't appear to do anything. A similar thing happened in my previous lab but I was at least able to confirm it mutated the gene I was interested in. Why does this keep happening?. A little bit of investigating revealed that all of the dodgy excisions I've made are ones that use the "Epgy2" P-element. It seems this element takes a perverse joy in screwing you over with confusing results. It also seems to have a tendency to lose the white gene without losing the P-element, so beware if you are doing an excision with it.
Honestly, I think it's time i try some of the new mutant making techniques. Not recombinational knockout as that seems like a ball-ache too. TALENs seems to have potential.

And that's the bad news for the week. I'll leave you on the cliffhanger of the good news until tomorrow (because I have stuff to do now).

Fire Drills

Fire drills are essential for large buildings but they can be a massive pain in the ass and potentially disastrous when researchers are "surprised" by a drill. If you are in the middle of a massive experiment that can't be put on hold or you have got that slot on the confocal you have waited a week to get then a half hour drill can really ruin things.

Valentine's day for Biologists

Being a scientist can be lonely, with little time for love - outside the love of science! To prove that we are still capable of emotion (and pattern recognition) here's a few pictures I found via a quick google search. In the true spirit of science I'll also tell you what they really are to dispel any undue sentimentality,

This one is taken from Helen Jacques and is a picture by Mathieu-Benoit Voisin and Doris Proebstl from London. "The researchers are studying how white blood cells move from the blood into into damaged tissue to cause inflammation; for example, after a heart attack. They were using using fluorescent pigments to stain two key players in this inflammatory process – pericyte cells from the blood vessel wall (stained red and blue) and collagen (green) – when looking through the microscope they noticed that the cells had arranged themselves into a heart shape"

This one is cheating a little but it's animated. You have to go to the web page to see it though.

This one is sweet...

That's the effect of tuberculosis in the lung of an infected mouse.

I guess the take home message is that scientists get bored when spending hours on a microscope and that horrible diseases can at least look romantic while they are infecting or killing you.

And let's not forget what the real thing looks like.

 Happy Valentine's day!

SILAC'S BACK! :)

For those of you who couldn't bare the cliffhanger of my last post, here is some good news - the cells seem to have clawed their way back and are now looking good for being confluent before the media is exhausted.

Phew.

Other decent news is that my antibody and the siRNA works. Ish. There's 3 bands, two of which disappear with siRNA but the strongest band not so much. Antibody for gene2 doesn't appear to work and instead looks like a dirty smear. This is the reason I didn't check the results until today. If I'd seen that at the weekend I would have felt the time spent there at the weekend was for nothing and have called it "bad" news. On a Monday I'm more prepared for things not working, hence this is "decent" news.

Are you confluent? Not nearly confluent enough.

On Friday I had to decide whether my "super-confluent" cells for my SILAC experiment were superconfluent. Unfortunately the lab "Aragorn" wasn't in to advise me and it looked like a do or die scenario. After consultation with other members of the fellowship lab it was agreed they were confluent enough and that they may die without more food. So I went ahead and split them.

I was in at the weekend and the king had returned. I told him about the scenario and he told me they weren't confluent enough and chances are the media will be used up before the cells are confluent again. As we are awaiting more SILAC media it could be the experiment dies.
"Aragorn" warned me; "A little more caution from you; that is no trinket you carry"

Now I just have to cross my fingers that those cells make an amazing comeback over the next 5 days.

Throwing the Baby out with the Bathwater

Started a SILAC experiment which i still maintain sounds like a great name for a villain, preferably a cyborg one. I've been told repeatedly that it's quite an expensive process so I was very focused on not screwing it up - so much so that I forgot to think about screwing up other things. Case in point I was splitting my cells, with gene-GFP stably expressed, into the SILAC medium and forgot to keep some of the cells for maintaining a line with.

Checked the -80 stocks and I didn't have any left so I'm going to have to reinfect the cells. Logic dictates it worked the first time so should work this time but experience dictates it probably wont be that straight forward.

The SILAC experiment remains on track for now though.

I'm still giving my flies the silent treatment due to their refusal to show a phenotype and for the P-element excision giving utterly bizarre PCR results that make no sense whatsoever. I'm starting to think the gene may be the gene for using quantum mechanics as it can only be observed indirectly as a null but never directly.

When Lab Colleagues Leave

A colleague from the lab left this week. He'd accumulated quite a lot of kit over the years and as he was tidying up on Thursday the rest of us began circling like vultures trying to find the prime pieces of meat for when he left. I actually cheated and asked him if I could have some of the boxes.
He officially left on Thursday and I think if there had been a time-lapse from 6pm Thursday to 11am Friday it would have looked a little bit like this (don't watch if you find decaying animals upsetting).

On one hand it feels a little disrespectful removing a person's stamp on the lab but on the other it's good that we recycle equipment and don't waste money buying new stuff all the time. The other perk is that there is a little more room for me to use now as I can use his bench to do stuff when mine is cluttered. The only downside is that the only other person in the bay area will also be leaving in a few more months and then I'll be all alone in my domain. Unless someone else joins the lab and then I'll have to start scent marking centrifuges, fridges and freezers.

Good luck to Colin, with whom I was able to share many silly scientific flights of fancy. I will remember him whenever I use his scissors, large Gilson and stopwatch.