Week 1 - 7th-11th June 2010: Plasmid DNA Purification...And A Lot More!

Firstly, I'd like to apologise for the extremely late second post. I promise to keep them more regular from now on.

So... I can't believe I've already spent two weeks in the lab... The time has just flown by and I've already learnt so much!

Today I thought I would tell you all about my first week in the lab; including how I'm finding working on my project and in the lab in general. I'd like to start off by saying how awesome it is working on this particular project. This is because, not only has it given me the opportunity to learn and carry out ample methods and techniques, but it is also fascinating! In addition, I'd like to add how awesome the atmosphere and the people in the lab are too and well... it's just great!

I began my placement on Monday 7th June, by attending the weekly lab meeting (which entailed giving and watching/listening to presentations given by members of the lab). I find the lab meetings a great experience for learning about break-through research, related to your field of work, and a chance to brush-up on some of those presentation skills.

Following the meeting, I was provided with some background information on my protein (Pds5A). Luckily... this all made sense as I had already done some reading around the subject (thanks to Dr Patel). I was told that I would be localizing a truncated (that means a chopped protein) and full length version of Pds5A and that I would start the week by purifying a plasmid construct, composed of Pds5A DNA attached to DNA of a red fluorophore. I was glad - because I was going to get stuck into the fun stuff straight away! I was given full guidance, by Dr Patel, when purifying my plasmid and the Masters and PhD students were a massive help. They helped if I ever got stuck and they also made sure I knew where things were kept, when I forgot (well... the lab is a big place with lots of chemicals and equipment). They instantly made me feel welcome in the lab which made me feel at ease - something I was weary of before I started.

By Thursday evening I had purified my plasmid DNA and carried out electrophoresis to confirm that I had purified the correct Pds5A DNA.

I also split my own set of HeLa cells which I transferred to coverslips allowing me to transfect them with the Pds5A DNA on Friday.

Thus, I did a lot in my first week allowing me to learn and carry out some new techniques, using equipment that I have never used before. Something I found really amazing was how easy it was to perform and understand these techniques, after learning about them over the past two years.

I really, really enjoyed my first week and by the end of it, I feel I was able to find and do many things independently and think more logically in general. Luckily... nothing went wrong all week!

I should be posting more on my second week in the lab over the next couple of days, so please keep a look out.

Until then take care,

Karishma

Hi! Welcome to my first ever blog!

Firstly, you may be asking yourselves...

Who Am I? And for What Reason Am I Writing This Blog?

Well... My name is Karishma Asiani and I am second year undergraduate at the University of Leicester; studying BSc Medical Biochemistry. As a university student, after completing my exams, I am given a (well deserved) four month break. Now, instead of me wasting this time watching TV and socialising with friends, I decided it would be a great idea to gain some good laboratory experience. Not only would this prove to be beneficial for acquiring good laboratory practice (which would help in my final year dissertation) but, it would also allow me to make a choice on if I can see myself obtaining a future career as a scientist, working full-time in a laboratory based environment.

So... Thanks to Dr Raj Patel, I have been extremely fortunate to attain a Summer Vacation Studentship, funded by the Biochemical Society. I am currently working on my project.

What is My Project Based on?

I began working in Dr Raj Patel’s lab, at the University of Leicester, on Monday the 7th of June 2010. Here I am going to be examining the intracellular localization of Pds5A in the mammalian cell, with the use of live cell imaging.

Pds5A is thought to be a sister chromatid cohesion protein, that regulates the maintenance of cohesin - the protein complex which is responsible for binding sister chromatids during the S phase, G2 phase and into M phase of the cell cycle. Cohesin is composed of four core components, which are: Smc1, Smc3, Scc1 and Scc3 (SA1 and SA2 in vertebrates). These have been suggested to form a ring-like structure to hold the sister chromatids together until the Scc1 subunit is cleaved by the protease separase during mitotic metaphase, allowing sister chromatid separation and the onset of anaphase.

Correct chromosome segregation during mitosis is vital for maintaining genome integrity in eukaryotic cells. Errors in the process can result in aneuploidy and ultimately lead to the development of cancer or birth defects.

The localization of Pds5A should therefore permit us to deduce and understand the intracellular mechanism and role of Pds5A, in regulating sister chromatid cohesion. Further analysis of these biochemical events should increase our understanding of Pds5A hence, aid in the development of methods of treatment against cancer, birth defects and targeted diseases caused by defects in the protein.

Project Aims and Objectives...

The aim of this project is to generate a GFP-Pds5A expression construct, that will be transfected into HeLa cells. The cells expressing the chimaeric protein will then be monitored using live cell imaging; allowing the determination of the intracellular localization of Pds5A and thus its mechanism of action.

Blog Objectives...

I aim to make regular posts on this blog, every 2-3 days, throughout this eight week placement. They will include things like: the different techniques that I use, the results that I obtain and (more likely than not) the things that will go wrong (*keeping fingers crossed for that not to happen too often*)!

I am hoping that this blog will prove to be useful to read for other students interested in carrying out a summer vacation project, researchers and scientists interested in this discipline of cell biology and my supervisor (Dr Patel).

I would like to thank you for taking the time to read my introduction and would also like to make a request, for you to subscribe to my blog so that you can stay up-to-date. I will try and keep my posts as short and sweet as possible from now on and I hope you enjoy reading them!

Take care and have a nice day,

Karishma