Tuesday, 5 August 2014

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!!!


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