FIVE AND SIX

Here's some piggy choromosomes - cuties.

(I snapchatted them)

So I thought I’d join up these last two weeks, mostly because Week 5 was pretty much a repeat of the previous week (and, like in Week 4, the probes didn’t work – trying them again this week so fingers crossed!). Mini excursions of the last two weeks include: taking a nice little trip up to the Tissue Culture lab for some nanodropping for the Genomiphi (very fancy with their double doors and lots of light) and also going on a lovely expedition to stores to stock up on stock-making stuff which was fun :P.

Pam Genomiphi-ing

Two loads of ovaries have arrived and hopefully a good bit of fertilisation occurs (so that I get to play with the micromanipularator more). Managed to do quite a bit of embryo biopsy these last two weeks which was exciting as ever (and still requires me to go “pew pew” everytime I use the laser (and slightly disturb Evi in the process.

Cooperative little blastocyst coming out of the hole I just blasted

These little spermies didn't want to leave

Evi’s been busy writing up her Masters thesis so I’ve been doing some of the fiddly IVFy bits that she normally does like getting all the cumulous cells off of the (maybe-)zygotes. Did lots of media-making again the last two weeks (definite Media Queen) and the pH meter and myself are starting to accept one another, which is lovely. Last week I found a new favourite job (ok, slight exaggeration) – making stock. Mixing up powders and water and then aliquoting the stuff into hundreds of little eppendorfs and then writing on them really teeny-tinily is apparently my idea of a good time, who knew. Seriously relaxing, who needs spas? I managed to leave the freezer slightly open (everything was still frozen, as were the doors…shut.) so had to go on a little expedition to find homes for everything that was in there so that the thing could be defrosted (and then had to search for stuff while making the rest of the media. What a mission.) Silly me!

Evi posing with "milkshake" (i.e. Piggy sperm)

To get the oocytes out of the ovaries we use this fancy aspirator (hoover with a needle). One of my jobs this week was to figure out how much difference using the different settings makes as well as if the different gauge needles actually have much of an effect on the overall suckage. Has been great telling people that I spent the day “weighing water” – science J.

Spending the end of the week filling out forms and results before going to V-Fest, woohoo!

WEEK 4

Started off the week a bit differently by heading up to the tissue culture lab to do some nanodropping. Basically, there’s this really fancy bit of kit where you drop a bit of your DNA sample onto a tiny glass bubble and bish bash bosh a posh graph appears telling you how much DNA you’ve actually got in there (thanks to different wavelengths of light and stuff).  Once this bit was done we headed back to the lab to get on with the amplification. The aim here is that I want to make a probe for the piggy chromosomes to be able to help in figuring out (using FISH as opposed to PCR) whether it’s a boy or a girly pig. All the stuff needed to do this comes in a pretty box and everything is nicely organised and labelled but, of course, that didn’t stop us from accidently using the wrong buffer for a couple of steps (our bad) – didn’t seem to matter too much (not that mine worked ANYWAY) so that’s handy to know!

Mixing stuff and spinning stuff

Next up is Nick Translation which cuts up your DNA and replaces little bits of it with tagged nucleotides to allow for the labelling (the whole point in this thing).  The nicely nick tanslationified samples are run on an agarose gel to check to make sure the DNA was cut up nicely (and to see if you’ve just done it plain wrong) before you purify the probe. If the bits have been cut too little there’ll be lots of unspecific binding, if they’re too big they wont hybridize correctly..you want them juuuuuuuust right. To see the banding you have to put the gel block in this fancy light machine and it looks all abracadabra-y and then shows you a pretty picture of the banding J. What you want is a smear to show that your sample has been cut up into different sizes as opposed to seeing one clear band which would mean that nothing happened.

Gel making and fancy machine working

To see if the probe has worked you need to put some piggy chromosomes and probe on a slide and do some FISH so that you can actually see what’s going on. One of my samples stopped working halfway through the process while the other one waited until the end to decide it didn’t want to cooperate, but that’s cooool, try again next week! Practise makes perfect ;) What you’re meant to see when you go on into the dark cold microscope room (where I spent most of last summer) is little sparkly green lit up bits on the chromosomes (where the probe has stuck to what you’re looking for) whereas I just had a bit of a green speckly mess.

The actual piggy IVf-y bit of my project continued with another batch of beautiful ovaries (which arrived a bit cold so felt foul – and a couple of them looked a bit diseased which was fab). As per usual, the eggs were moved to some different media to make them grow nicely on Tuesday (using the cute little EZ grip pipettes and LOTS of concentration) and then on Wednesday we lit some candles for fertilisation ;).  Before they could have some fun though we had to do more pHing (yey.) and a fair bit of moving eggs from nunc plate to nunc plate. The sperm had to be prepared (bit more effort than just a pep talk) by making them compete in the swim-up tubes. Some of last weeks eggies and spermies did some good work and we had some cleavage and even a morula, yipeeee!!!

3RD WEEK

Another week upon my quest to become the Media Queen! (It’s actually going quite well). Started out the week by banging out a few Falcons of Piggy IVM media not only faster than before, but remembering that they live in the fridge (as opposed to the incubator) and end up in Nunc plates, making themselves useful (instead of being chucked in the bin). YEY! The incubator we have been using decided it wanted to break so kept making a joyous noise (see video) which probably just put me off going anywhere near it! The most fantastic bit about the IVF lab is that the pig stuff doesn’t like aircon..fabulous when it’s almost 100,000 degrees thanks to the heatwave!

Silly incubator 

This week was a big ovary week with a nice(?) flaskfull arriving on Monday and a few more (not as many as expected L) coming on Wednesday! On Monday afternoon I spent 2.5/3 hours sucking oocytes out of about 70 ovaries (delicious) but on Wednesday had to stay at home thanks to a bit of food poisoning (could have been interesting trying to aspirate stinky pig ovaries in that state).

Piggy ovaries in a jar, piggy ovaries in my hand and piggy oocytes and goo.

Towards the end of the week I started getting things ready for some mini-prepping and labelling. First step was making up a couple of plates (haven’t done that in a while so had absolutely no clue what I was doing although I definitely should have! Bad scientist L) which were then streaked with BAC clones and incubated overnight. The BAC clones (called Pig-E BAC…piggy BACK..piggy-back, gerrit? tehe) are stored in an extremely cold freezer which makes it feel like you’ve stepped into the arctic when you open the door – which oozes a whole load of condensation/fog (pretty epic)!

Frozen freezer

In an attempt to fix the extremely annoying incubator the CO2 canister was changed to see if that’s what it was moaning about. Kate and I had the fun task (I say I but I was just there for support and door opening ;) ) of going to stores to get a new one. This is the furthest lab away from stores, CO2 canisters are heavy and their trolleys are a bit pants. Managed to drag it back and get it refitted just to find out that the canister wasn’t the problem at all.

Fertilisation Friday came about really fast! The morning was occupied with exercising some sperm while separating the “boys from the men” using a swim-up tube which is pretty much a spermy race. Some pretty photos were taken of the nicely matured eggs (I’ll put them up next week) and then we all headed off to Pizza Express for Kara’s birthday lunch J (she’s nearly finished her PhD, woohoo!). A full (both through busyness and food) afternoon was compiled of fixing and mini-prepping. We fixed some embryos (stuck them to a slide) so that we can do some FISH on them to test our sexing stuff (not as naughty as it sounds) however, apparently this rarely actually works- I was told the embryos “float off into the sky” but I think that was a fib. The mini-prepping was done to get ready to make the probes for the FISHing (busy busy busy).

Nicely mini-prepped and ready for next week!

  

WEEK 2

What a busy busy week! (But also a really cut up and inbetweeny one). No ovaries this week because of the lab away days but there are loads coming next week so a lot of my time was spent making media (or cacking it up as the case may be) and learning a bit about PCR! I also had all of my old housemates staying with me so they could graduate (don’t leave me!) CONGRATS TO THEM ALL!

So, at the start of the week I went about learning what the PCR fragment analyser actually is/does (with the help of Evi and her notes, obviously). Basically, what this massive noisy machine does it estimate the size and amounts of DNA fragments as well as the alleles that they represent. What we’re using it for is to see if we can determine the sex of a pig embryo based on the presence of a specific gene.  To get it going you have to pull out loads of fragile looking things and replace them with even fragilerer things! You then load your samples and a ladder which you’ve previously PCR’d and voila, after a few hours you’ll get some nice graphs showing you what was inside.

Why so sad Beckman? (Kinda looks like a sad/happy snowman)

Making the media was a fun experience…it was all going fantastic just chilling in the IVF lab with no aircon (the piggies don’t like it) during some of the hottest days ever until it got to the bits where I needed to measure the pH of the stuff. Stupid pH machine absolutely hates me (yes, that is possible) and it took an age for it to decide to tell me what I wanted (this is after 3 people recalibrating it!). If it wasn’t so useful and expensive I would’ve hurt it but instead I just gave it a piece of my mind and moved on. Naughty pH meter. To top it all off, clever Daniella over here thought she knew what she was doing so went straight ahead and put all of the media she’d spent the last few hours straight into the incubator…it lives in the fridge…D’OH. And that day’s life lesson was that Daniella knows nothing. I joke, it’s to thoroughly read instructions.

In the bin!

On Friday I spent the day remaking everything (the silly pH meter lied to me and forced me to have to remake one or two agaaaain) but practise makes perfect and I WILL become the Media Queen.  The media are now all nicely resting in the fridge and are ready and waiting for their time to shine when the ovaries are delivered next week (Woohoo more hoovering)!  

I hate you.

The lab away days were fantastic! A great opportunity to get out of the lab and discuss what’s been going on, future plans and, of course, do some “team-building” (also known as a fantastic excuse to play ridiculous games like blindfolded maze and the box game). It was crazy sunny and absolutely beautiful. It was great seeing everyone a bit more relaxed than their usual crazy lab-selves and I’m very grateful to have been invited along! 

Just down the road from the Lab Away Day, so puuuurdy!