Friday 22 July 2011

Agarose, agarose and more agarose...

It has come to the end of my first week (I know I only did my introduction post yesterday, but y'know, time gets away from you when you're in the lab)-- and I've found myself to be quite the little busy soul.

On Wednesday, which ended up being my first day due to complications getting back from Spain, I was eased in to the lab environment with some bioinformatics work. Normally I'm not one for bioinformatics because it winds me up (I get stressed out in bioinformatic practicals like you wouldn't even believe); but I actually found this particular bioinformatics work pretty enjoyable. Probably because instead of simply database trawling, I was analysing the latest CHiP data that the lab had received back.

The data that I was given outlined points in which Zta (a DNA binding protein) is found to bind DNA (discovered experimentally by one of the PhD students in the lab). The specific binding regions were then mapped against the human genome to give two different possible data outcomes: unique peaks and repeat peaks.

It was my job to identify which of the peaks were unique-- which were then placed in a separate file which is going to be further analysed in due course (probably next week). The idea is for me to identify the unique peaks that have the least single nucleotide polymorphisms (SNP's) and that correspond most closely to the transcriptional domain-- ultimately for us to design a primer pair which can then be used to identify Zta binding domains.

Wednesday afternoon was my health and safety talk with the added addition of the lab tour. With the lab being split between three separate research groups, things are in very specific places so I've had to learn where to find all our equipment opposed to using anyone elses' by mistake. I think I know where most of the equipment is now (I've drawn myself a map) but only time will tell whether I will be able to remember how to read my map when it's not so fresh in my mind.

Anyway, I've been set up with my own little work space within the lab, although at the moment I tend to spend more time at Angelica's bench (the post-grad who is showing me the DNA ladders), just because I'm not quite ready to fly completely solo yet. Having my own work bench though sort of makes me feel all official and properly scientific.


Thursday was spent learning the ins and outs of Polymerase Chain Reactions (PCR) and Agarose Gel Electrophoresis. In total the experiment has 4 fragments of DNA that we're trying to analyse; all of which are overlapping (1-4)-- in our PCR we took fragment 1 and 2 and amplified them which, well, was a little bit of a fail because when we analysed it in the gel electrophoresis only fragment 2 presented and even then it wasn't particularly amplified.

I guess though, that's what happens with science. You're experimenting. You're not always going to get the results you want (although, for my first PCR I would've liked a little encouragement that I was doing something right!)

In the gel electrophoresis we were testing fragment 3 and 4 on an extremely large scale-- so we ended up making giant agarose gels for them to run on. I didn't even know they existed in this sort of size! The idea of this experiment was to determine that the two fragments were of different sizes and no cross-contamination had occurred as well as providing us with an isolated DNA sample that we could then purify.

As it happens, this worked extremely well. We ended up running 3 separate gels which all showed the desired results which just helped to solidfy our conclusions (one gel with just sample 3, one with just sample 4 and one with sample 3 and 4 side-by-side):
  Sample 3 and 4 side-by-side gel 
(sorry about the quality, it's off my phone opposed to scanned)

After running the gel, I then had a brief experience of feeling a little like a member of the CSI team. We had to take the sample up to the UV room to cut the agarose regions that contained DNA out-- which obviously means full protective gear including the face-shields (hence the CSI comparison, it does make sense). Nervous doesn't even begin to cover what I was feeling faced with UV-- I guess that because it gets drummed into your skull how dangerous it is, when faced with it I was more than a little bit wary. Of course, there is no need to be really if you're careful, so separating the samples from the agarose came off without a hitch.

Today I was taught a method of gel extraction which would allow us to purify the DNA and separate it from the agarose gel. The procedure was pretty easy to get your head around (everything came in a handy little separation kit, which makes life that little bit easier). It bascially consists of dissolving the agarose gel in a buffer and then centrifugating the resulting mixture to separate the two elements.

We then ran the resultant elements of sample 3 and 4 against already purified samples of 3 and 4 (done in a previous experiment prior to me arriving) on a small agarose gel and got absolutely perfect results:
Ladder, Purified 3, New 3, Purified 4 and New 4

This means that on Monday we can set up Western Blot for sample 3 and 4-- which although it seems like it's going to take a long time, looks like it should give exactly the results we're aiming for.

Theoretically of course.

Thursday 21 July 2011

Hello lab...

My placement has finally started!

It feels like I've been waiting for my summer studentship forever. In reality I've only really known that I'd be definitely be doing one since April, even if I had spent from September onwards trying to get one.

The lab that I'm working in is located in Sussex University (in Brighton, just to complicate things). It's the very same university that I'm studying Molecular Medicine at, currently between my second and final year. Ultimately my aim is to go in to medical research, or perhaps even become a clinical bioscientist... All I know is that lab-work is where I'm bound in the end.

The lab that I'm actually working in is in itself is split between three different research teams; two working on Epstein Barr Virus and one working on S. Pombe yeast. I'm lucky enough to have found myself a place on one of the EBV teams working with Dr. Alison Sinclair and the rest of her research team-- all of which are extremely lovely and have been nothing but welcoming since I arrived.

My project is specifically focusing on trying to identify the origin of lytic replication (OriLyt) of EBV. Epstein Barr Virus is one of the most common human viruses; with over 90% of the population infected. The virus itself normally infects an individual at a young age and will remain latent and asymptomatic. It becomes a problem within the population in one of two situations:
  1. When the individual contracts EBV in later childhood/early adulthood they will develop mononucleosis (also referred to as mono)
  2. In some patients, the EBV can cause the development of Burkitt's lymphoma (cancer).
Ultimately the aim of the research is to allow us to understand the virus on a higher level-- and maybe one day allow us to identify a way of preventing the virus from infecting individuals and hopefully irradiate virally-triggered Burkitt's lymphoma. I may be a little optimistic and unrealistic when I say that I hope the research I conduct over the next 8 weeks helps in some way.

Anyway, this project is ongoing and it has been passed on from post-grad to post-grad with each person making their own discoveries and furthering the research. It's finally fallen in to my lap, so I'm just hoping I can prove to be as good as the post-grads. And I hope what I end up discovering will be of interest and relevance to the next person.

I think that's probably enough of an introduction for now. I will try and update tomorrow after I've finished the lab to give a run down of the actual experimental side of life. All I will say now is that I'm so very glad I've got this opportunity.

Natt