Does anyone else smell burning?

Hmmm it's been a little while since I last posted hasn't it!! Mainly because the microscope was fixed just before I went off to MIF and therefore it was hell for leather for the next few weeks as I attempted to do all the work I haven't been able to previously. On top of this I was still trying to do all my westerns and write that darn review (hated HATED review!).

But as we know... this microscope - it is cursed, as the lightbox blew again last Thursday! Luckily it's nothing serious and should be back in action sometime this week we hope (and pray).

Last two days have been relatively long and unproductive especially in the review stakes. I have written the first draft and now have to correct and add in a few additional sections but UGH, I just don't have the ENERGY. I think I've hit that mid-year wall and I know I'm not the only one. Luckily for me I have some conferences (aka holidays) so hopefully I can come back nice and refreshed and ready for the rest of the year... unfortunately for me my review is due before that. The big boss, AC, is actually away for this week to review grants, so I need to have that second draft ready for the beginning of next week... so far I've written two additional paragraphs... So I need to pull my socks up and start writing and editing... which is totally why I'm writing this entry instead :)

Yesterday I almost burnt the lab down.... okay... this is a slight exaggeration but I did burn foam and melt plastic (and just to clarify so I don't come across as some crazy pyro, it was by accident!). I needed to quickly boil some enzyme to deactivate it* so I set up the tripod and beaker and turned on the bunsen burner. The bunsen burner that I used is CRAZY!! (Actually I originally thought it was the gas tap that was crazy - but after a lil experimentation this afternoon we have determined it is the bunsen burner!) Why is it crazy? It's got an amazingly strong flame with is seriously like a metre in height!! Like no joke - it's freaking dangerous!

Look at it there - all high and mighty... it's bloody taller than the selves.. and this is on the low nice flame instead of the 'instant 3rd degree burns' bright blue setting!! However it was the bright blue setting that I put under my beaker, with my lovely lil floatie** holding my eppendorf tube which was closed by a lovely bright pink plastic tube holder. I normally boil my enzymes for about 10 minutes, mainly cause it seems like long enough for all the damage to be done to my protein. However around bout 3 minutes in, a curious smell is starting to reach my nostrils... I wasn't certain what it was but it did smell a lil like burning... something somewhere is burning...

Uh Oh.

I approach my beaker and notice the lovely green floatie is very quickly going a lovely shade of black and my beautiful pink tube holder is melting... MELTING!! At this point I turned the gas down...

And here it is... my burnt and melted monstrosity. (Please note... it did not look this originally!)

The worse thing is - when the lid closer decided to go all gooey on me it manage to leaver the lid open meaning a whole bunch (well a few microlitres*** actually) which means it's useless and I have to throw it away... farewell $115.00 dollars... I shall miss you.

On top of this I totally almost killed someone the other day coming out of the cold room. First off I would like to state that this WAS NOT completely my fault.

The cold room has a big heavy door on it - obviously to keep the place close - and you really need to give it some heave-ho to get it moving... even if you are as muscley as myself... *cough*. However, the micro labs have put two freezer right outside the door. I went in and close the door firmly behind me as I put some stuff away. As I went to leave I opened the door with a bit of force RIGHT INTO the honours student standing there grabbing stuff from the freezer... she was not permanently injured and I was appropriately apologetic... however now when she sees me she has a tendency to make a door joke... awkward...


* BCHM101 - Enzymes (well all proteins actually) need to be 'folded' in the correct shape for them to be activate and carry out their function. High temperatures cause proteins to denature (which is just a fancy way to say unfold), which is why boiling an enzyme deactivates it.

** Floatie - a highly technical term for pieces of foam with holes in them which allow you to float your precious eppendorf tubes in water without the lids getting wet.

*** One millionth of a litre, or one thousandth of a millilitre.

“If I'd known how much packing I'd have to do, I'd have run again.”

I hate packing.

Even when I'm just packing for a weekend away I always end up feeling uneasy... just a lil queasiness in the tummy.... and it's always telling me that I've forgotten something. Even when I've been through my mental check list a hundred times

Undies ................. check
PJs ....................... check
Clothes ................ check
Toothbrush ......... check


I still think there is something, something important, that I'm missing! I don't know why. However once I'm at my destination the feeling passes and I go on living my life in happy blissfulness.

I had to repack for MIF last night. I had all my stuff in a relatively large (& heavy) sports bag however had then discovered we may be staying in shared dormitories(!!) where I would need to lock up my bag and also that I apparently wasn't going to be able to park on site... which means I'd need to lug my bags from the car to the school. So I pulled out Mum's suitcase... it's lockable and has wheels!! It's also the smallest suitcase in the house... though it was actually a lot bigger than I remember. I look like I've packed for a few weeks instead of a few days! I still feel like I've forgotten something (not sure what!) but I also feel like I've overpacked as well... there is no winning with my brain.

Last night I also received acceptance for my ANZBMS abstract. However... this paragraph:

"Your work has been highly valued by this stringent peer review system. Since your work is expected to receive a lot of attention at the scientific meeting, the ANZBMS Council asks that you take considerable care with its presentation."

SCARES ME!

I'm hoping that it comes standard with every abstract acceptance! (I'm waiting for Mahvash to get hers!)

Busy... yet not

So it's been kinda of a weird time in the lab recently - while my microscope is STILL out of commission I have been busy with lots of molecular biology/western blotting stuff. So it hasn't been a total holiday but it has been quieter than normal.

We've been trying very hard to get the Gateway Cloning system up and running but we have run into a few problems - mainly getting our AttB PCR amplifications to work. We've redesigned so of the primers so hopefully that'll work. When I did actually get them to work, VA (our new postdoc) accidentally told me the wrong antibiotic to grow our transformed colonies... so as you can imagine that wasn't very successful! But hopefully once the new primers arrive it'll be onwards and upwards.

Thursday and Friday of last week were pretty hectic as I discovered I had to write an abstract for the ANZBMS conference by Sunday. I spent most of Thursday hurriedly crunching numbers so I could write it up on Friday - however all was in vain as they extended the deadline till next Monday! But at least it's now out of the way. It's currently with AC and I'm sure I'll be receiving my first round of revisions very soon!

The other exciting thing I've been doing is organising my ASBMR conference trip in September. The conference is in Montreal, Canada which is very exciting. I already have my accommodation organised and I'm planning on booking my flights by the end of this week. I discovered that it's going to be cheaper to fly home via New York... which I will definitely be taking advantage of! I've put a reservation on a hotel room in NYC for 3 nights, which is very exciting, hopefully I can
get to visit a lot of famous landmarks while I'm over there. I'm also hoping that I can see a show on Broadway... I really really really want to see Avenue Q - so I may book a ticket in a month or so to go see it while I'm over there.

What's that smell!

B-mercaptoethanol smells like crap...

Now the lab smells like it... opps...

Bloody FRETing Red Tape

So more microscope related lab woes... As a lab we are hoping to get into some FRET work. What is FRET? Well, FRET stands for Fluorescent Resonance Energy Transfer and refers to an energy transfer mechanism between two chromophores (or molecules that are responsible for colour. In FRET you have a donor chromophore and an acceptor, when they are in close approximation the donor in its excited state is able to transfer energy to the acceptor (as seen in the figure to the left).

The newly emitted light from the acceptor is different from the donor - leading to a change in the fluorescent spectra which we can measure.

We are hoping to use FRET to see conformational changes in our receptor of interest. By inserting CFP and YFP into different sections of the receptor, we are hoping to measure how FRET differs when receptor is bound to it's various ligands. Do certain sections of the protein move closer or further away from each other to allow ligands to bind? Do they move differently for different ligands? Well... hopefully FRET will allow us to work that out... however for us to do FRET in our lab is requires us to buy new filters for our (probably cursed) miscroscope.

Somehow I managed to be put in charge of buying these filters. Mainly because I require them to try a new FRET based method of measuring intracellular calcium for my STC-1 cells (which don't seem to like Fura-2AM all that much).

So I email "The Company" asking whether our microscope was capable of doing this, what new equipment would we need and (most importantly) how much was it all going to cost. The next day one of "The Company's" reps just happened to be in the building, so they came around, we had a chat and he said that I'd get a quote in the next week.

A week went by....

Two weeks went by... at this stage I'm thinking that our rep has forgotten us so I send off an email. Turns out they had been trying to contact us, but by phone as the rep wanted us to talk to the product manager to make sure we were getting the right filters etc. I ring the product manager while he's in the middle of a conference... but eventually we get in touch, talk about what we need and we finally receive our quote... (with a special 'sorry its taken so long' discount)

The quote offers High Efficiency and Standard Filter Sets... AC (the boss) isn't sure which one we need... it also doesn't list excitation filter holders which we require... so after a few phone calls later we have a revised quote and have (finally) decided what we are getting. Yay! I send an email to 'The Company' and we wait....

After hearing nothing after a week I email our rep who tells me they haven't received an order yet. Puzzled I start to ponder... and then realise that maybe I was meant to put the order through downstairs via our store/purchasing department. (Opps! My Bad!)

The entire order equals up to ~$4,900 containing our filter set (~$4,600) and filter holders (~$300). The next day my one of our postdocs RM comes back to me with the order form. Apparently it had gone over the purchasing department threshold and they couldn't put it through!

So I went down and talked to the store guy who explained that it was over the $5,000 threshold.

I'm Sorry... did I hear that wrong... My order was for less than $5,000... Oh what? Once I add 10% GST ... Ah... yes I see...

So Mr Storeman tells me that I eitehr need to go around and find and cheaper quote (impossible as "The Company" is the only supplier) or get them to write a letter on the company letterhead stating that they are the only supplier for Australia... and hopefully the purchasing department will process it all in about a month or so... (ANOTHER MONTH! ARGGHHH!!)

I decided to have a quick chat to the Boss about it... I was secretly hoping that, as he also doubles as Head of School ,he may have the sway to get this order through without having to try and get this letter... He firsts suggest we split up the order into two, which allows the filter holders to be bought however the filter set is still over once GST is added (by $60!). He also chats to Mr Storeman and brings up the fact that we don't actually have to pay GST on equipment... however it doesn't matter. Letter is still required.

Manage to get into contact with our Rep and explain the situation. After much complaining on the stupidity of the purchasing department by both of us, the rep tells me just to further discount the product so that is = $4,999.00 once GST is added!

Change the price and head down to the store... Explain what I'd done to Mr Storeman, who gives me a very uncertain eye and says "I'll see what I can do."

I haven't heard back yet.... *fingers crossed*

An Article of Substance

With the obligatory "welcome" message out of the way I thought I should probably write a proper journal entry...

At the moment I'm just sort of waiting around the office for our weekly lab meeting to start (in about 2 hours). It's been pretty bludgy for me over the past few week - which is both a good and bad thing. Obviously I would like to be doing more work, however, due to circumstances out of my control (i.e. Our fluorescent microscope being out of order) I've only really been able to do a few other things. I'm seriously hoping for the scope to back and running in the next week or two... so once that happens hopefully it'll be GO GO GO on the experimental front. BUT in all honesty... I'm beginning to think that this microscope is cursed!

First off, our 'waterproof' microscope got liquid all the way through it causing the reflector turret to stop moving automatically. We discovered that it wasn't the turret that was causing the problem... but probably the motherboard. Then the lens off the 63x objective managed to come unglued and could only be replaced by a 40x objective. Then the bulb died on the DG-4 wavelength switcher. When the new bulb was installed, the length of the main connector was changed meaning the light reaching the microscope was more than halved... but we didn't discover that until 3 months of crappy results. Now... it seems as if the bulb of the DG-4 has gone again and since we have no spares we've had to order some in from the US. While this allowed "the company" to take the scope away to fix the turret/motherboard problem... and apparently they manage to break something while transporting it back into the lab. *sighs*

Fingers crossed that this is the last problem we have for a long, long, LONG time.

Hmmm I've just looked out the windows and realised it is looking very dark and gloomy at the moment... not good. Tonight, I am (hopefully) going out for dinner with 'The Girls' (and yes I capitalised that on purpose!), however apparently our booking at the Flying Fajita Sisters is for the outside courtyard and I fear dinner may be cancelled if the weather worsens... which I sincerely hope does not happen as I haven't seen a few of them for a few weeks and I'm really looking forward to a good catch up!

Lift Agony

Lift's are out and stuck on level 2 again.. We seem to be having lots of problems with our lifts lately - in fact we've already had two academics and a few students stuck in lifts just today... I have a feeling it may be a plot to get us to all burn some kilos... It's days like today when I'm glad our lab moved down from level 7 to level 5 :)

Oh Happy Day

Ahhh I am so happy I could sing, jump, and dance a happy dance!! For the last 3 months of so I've been getting crap crappity crap microscope data.... considering this microscope technique is the main functional analysis I do having crappy data is really depressing because I can't prove ANYTHING without functional analysis.

Functional Analysis = IMPORTANT!!!!

But today... yes today we managed to fix it! AC came into the microscope room with me today and thought the light source seemed rather lack luster and started talking about the light box and whether maybe, just maybe, the globe had gone and no one had noticed.

This jogged my memory as I realised.... well... no that couldn't be the case as we had changed the globe recently... if fact we changed it three months ago. Around the time everything went up shit creek! This made us think at first that the wrong globe had been put in and that the globe just wasn't powerful enough. Quick email to our resident microscope Mr. Fix-it who comes down and has a look at it.

Turns out - when he changed the globe to first time he didn't realise that the main connector from light box to microscope must be a certain length for the maximum amount of light to reach the stage! Ugh!

3 months of useless data... all because some connector was not the right length... am I annoyed? Slightly... but in all honesty I'm more relieved than anything! Finally I'll be able to get some proper results!! YAY!

Also... tested my site direct mutagensis colonies... Actually the first colony I sent away for sequencing ended up only showing parental DNA... :( Luckily RC encouraged me to send off the rest of the colonies for sequencing just in case - and all 5 of them ended up having the mutation I wanted and 4 of them showed extremely strong sequence alignment to the reference sequence! So on to mutation number 2!

New Year, New Post

It's only the 8th of January BUT I'm already back in the lab (Day 2!) - tidying up my lab book entries from last year (I'm up to the 20/12... so very close!) and setting up some experiments for later on this week. Tomorrow I have our Summer scholarship student coming in to start her 'project'. It's a bit strange actually teaching someone else how to do things in the lab. I'm not entirely certain that I'm a great or even good teacher and half the time I still feel like I have NO idea what I'm doing in the lab... yet now I'm in a position of seniority! It's a scary thought!

Not that I haven't learnt a lot in the past year... I've had some definite highlights including winning an ASBMB Poster Prize @ Combio, having a paper published with my name on it (yay!) and swanning around Queenstown on the pretense of 'conferencing' @ ANZBMS. I've also learnt some hard lessons - backing up computer files is VERY VERY VERY VERY important... & nothing is EVER as simple as it seems!!

Today I've had a pretty frustrating day actually... I seem to be missing some of my data from last year - I'm assuming i must of lost it when my laptop hard drive crash late last year (and me without my backups!), but I'd assume that I'd have some record of it somewhere... BUT apparently NOT! It's extremely disappointing as it was the best quality data I had for my Alanine Threshold data set - so now I'll have to calculate my AUCs with out it.

Though there is some good news - my site directed mutagensis seems to have colonies... I'm testing 6 colonies tomorrow! Fingers crossed that I've got what I need.

The Label is there for a REASON PEOPLE!

Over recent summer holidays my lab received an inducible expression system kit which I ordered before I went off to enjoy the sunshine. Unfortunately, who ever recieved it stored it in the normal freezer ... which would of been fine if there wasn't a GLYCEROL stock of the host strain required for transformations! So of course, by the time I went to thaw it out they were DEAD, and now I have to order new ones, which have to come in from o/s... so now I have to waste my time waiting for them to come in. Argh! Not Happy Jan!

Brennan, Serial Cell Killer

So... its my third practical week into my PhD, and I've hit my first snag (already!). Unfortunately for me, my HEK-CaR cells have decided mysteriously to up and die on me. I'm not sure why... normally when there is a contamination you can work out which one it is by just having a look under the microscope.

Fungal contamination is normally the easiest to see, as in advanced cases it can be seen with the naked eye. They normally look kinda whitish/yellowish and fluffy.

Bacterial contamination is a little harder, but can be seen under a high powered microscope as little black dots that move around the cells. Not a good sign. They also cause a sudden change to the pH of cell growth medium (and therefore the colour!) from redish/pink to yellow

Yeast contamination is also visible under high powered microscopy. Must admit I've never seen yeast contamination myself, however they look like small ovals that bud of from each other.

The hardest contamination to detect is mycoplasma, mainly because it cannot be detected by light microscopy. In fact, we dont even test for this in the lab! Thought it normally has to come from an infected cell source (which we dont have, so I doubt this will affect us!)

Argh! I am trying deperately to save my cells, I'm hoping tomorrow they'll be looking better... But i doubt it. And if they die I'll have to thaw out a new crop!

Darn I hate cell culture!