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!!!