Day 3

Today was going to be quite a short day in the lab because I only really had two important things to do in my experiment. The first thing i needed to do was to make replica plates of the culture plates I made the day before (the possibly transformed cells).

Replica plating is a way of transferring the colonies growing on one plate to a new plate. I needed to do this because now the cells have had time to grow and express thier resistance gene (if they took it up) and so they could now be transferred to a plate with the antibiotic in its medium. This will allow the selection process to take place over night in the incubator.

Once i'd done this I made up another culture of the wild type S. Pombe and put it in the incubator too. I did this just incase my first transformation failed so that I could do another one straight away.

After i'd done these things I decided to stay and watch some of the other people working in my lab to see what they were doing (if they didnt mind). I got to see another transformation (in E.coli thsi time) and also a method of retrieving pure protein from yeast via the TCA method.

Hopefully tommorow I should see some evidence of whether my experiment is still working, fingers crossed!

Day 2

Well its been another good day in the lab. I now know how to carry out a transformation experiment with the DNA I obtained yesterday (from the PCR experiment) into S.Pombe fission yeast. It involved alot of centrifugation to prepare the DNA from the culture grown over night into a solution suitable for addition to the cells. I then got to plate out the possibly (hopefully) transformed cells onto agar plates of normal growth medium to be left over night.

The selection process will begin tommrow once the cells that have taken up the DNA have had enough time to grow and start expressing thier new resistance.

I also got to see a presentation by a student working on a similar experiment while I was waiting for part of my experiment to run. It was interesting to hear about what he'd learned because it was on a similar subject to my own project. It was good to see what sort of level knowledge I could have on the subject in a few years if I stayed on after my BSc degree.

I'm off to write up some of the methods ive been shown so that I hopefully wont need help when i do the procedures again, so thats it for today :)

My first day

Hi again,

Now that i'm done telling you about my project I can tell you about my first day at work! I spent the first part of the day becoming familiar with the labs, people, where things were and what the project itself was about.

My first aim in this project is to change the selective marker in the strain of S.Pombe I will be using later on.

Today I was shown how to use the polymerase chain reaction (PCR) procedure to amplify the new marker needed to replace the old one. A large concentration of the marker is needed to give a greater chance of transformation in the yeast culture. I was shown how to create a working PCR mixture and how to use a nice PCR machine to carry out the temperature change steps for the type of polymerase being used.

I got to make an agarose gel for later use in gel electrophoresis. I made this so that I could tell whether the PCR had been successful. I ran the PCR product next to a lamda Ecori/HINDIII marker, this marker is used like a ruler so that the size of the PCR product can be determined by comparison.

I then was able to view the results of the gel electrophoresis under UV light and take the picture below! :D This picture confirmed that my PCR was successful as I got the correct size fragments at 1.5kb:



Lastly I was taught how to grow a culture of the wild type yeast in preparation for tommrows transformation!

I really really enjoyed today, i feel I have learned alot and it was really nice to have a good result on the first day, hopefully i'll keep it up. Though i'm sure when it comes to doing it all by myself it will be rather challenging!

Thats all for now :)

The Project

Hi there,

I thought i'd give a brief introduction to the project and why i'm doing it.

I'm working with Dr. Felicity Watts in the Sussex Centre for Genome Damage and Stability. I feel very lucky to have been given this placement by the Biochemical Society and the Genome Centre because it has given me a chance to experience what it is like to work in DNA stability and repair research which is what I believe I may want to do in the future.

The project I have been given to work on concerns the loss of heterozygosity (LOH) in chromosomes. This is where processes have caused mutations that cause a loss of function in both alleles of a gene. LOH often involves the removal of multiple genes and even whole chromatid arms. In diploid cells such as our own, we should have two copies of each gene and if one ceases to work the other can fill in for it. LOH removes this second copy making the likelyhood of cancer or other diseases much higher.

The centromere is extremely important in chromosome segregation and mutations occuring in the centromere repeats may be what causes this loss and chromosomal rearrangement. I will be studying the stability of the chromosome with mutations placed in different locations using a genetically engineered mini chromosome in the fission yeast; S. pombe.

Well thats all i'll say for now, lest my post become to essay-like. Should make a very interesting project though!