12
Sep
2011

I started the week by manually extracting DNA from 3 tissue samples. This involved leaving them in protease K for around 45 minutes which enabled the tissue to be broken down so the DNA could be accessed. The samples then had to be washed and centrifuged with various different washes and buffers in order for the DNA to be extracted. I had to be careful not to cause any cross contamination of these samples so had to open the tubes individually and handle them carefully. I am still waiting on the streptokinase enzyme and for it to be tested to make sure that it is suitable for use on the samples, so my project has been put on hold until this happens. To fill in my time again I have been helping on various projects running PCRs and gels for different people within the lab. This includes testing dogs blood who are involved in a study testing the competency of the cat flea, C.felis, in transmission of Bartonella henselae  from cats to dogs.

Our adventures

We have had a busy weekend this weekend and have jam packed it full of stuff. On Friday evening as it was the first Friday of the month the art galleries, restaurants and bars downtown were all open until late so we decided this would be a good opportunity to go downtown and have a look around. There was live music in some of the venues and we had the opportunity to have a look at some art, jewellery and ceramics made by local artists. After this we then went out for dinner to a Japanese/thai restaurant. On Saturday were supposed to be going to the cinema to see The Help but we got the viewing time wrong so this has had to be rearranged instead we had a look around the mall and went out for lunch. In the evening it was the first American football game of the season and we were lucky enough to have tickets. We went tailgating for a little while before the game. This is where people sit by their cars and listen to music, eat food and play games before the football game actually starts. It was a really good experience and something that would not happen at home. The game was really good; there was cheerleaders, a marching band, cannons when we scored and the atmosphere was amazing. Even at professional sports in the UK you would not have as much going on before and during the game and this was only a college game. In the end NC state won which was really good as otherwise we would have not been happy.

On Sunday, we went to the beach. Beth, from Jade’s lab offered to take us as her family has a boat so we were able to go to beaches that were more secluded. Unfortunately after we had been on the boat for half an hour the engine overheated so we had to get sea tow to take us back to the marina as the boat couldn’t go any further. Luckily, some of their friends who also have a boat and live by the beach  saved the day and took us out to cape lookout on their boat. We stopped off at the beach and went looking for shells and from the beach we saw a pod of wild dolphins swim past the beach which was lovely. We then took a smaller boat out to what was supposed to be Shark Island. However, we think that Hurricane Irene had washed the island away as it had been a sand bar so this was possible. We had a lovely day with Beth and her family and after a tiring day we were glad to have Monday off because of Labor day.

On Monday, we met up with Jade’s friend from home ,Shekinah, who has been travelling around America this summer and has now stopped off in North Carolina. We went to the mall although we had a little trouble getting there as we did not realise the buses did not run on labor day so had to get a lift from Jades flatmate instead. We went to the cheesecake factory for lunch and had a look around the shops, it was a lot more relaxing than the other days which was nice and it was good to see someone from home.

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12
Sep
2011

I am still waiting for the streptokinase enzyme to arrive so I can extract the DNA from the rest of my samples. We did try placing the samples in the water bath to see if they would lyse as this had previously worked with the others.  However, unfortunately this did not work so I am having to wait until the enzyme arrives before I can continue with the project.

This week I set up PCR and running gels for various other projects and scientists within the lab as they are all extremely busy with their respective projects.

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12
Sep
2011

This week I have continued with my project testing dogs blood from Canada for Bartonella. In total I have extracted DNA from 32 samples , so I went on to perform PCR’s and gel electrophoresis on these samples.  From these samples 10 showed positive results so these were kept in order to be sent for sequencing. 

The samples which had previously been shown to be positive and had been sent for sequencing came back as failed sequencing which meant that the DNA that produced the band on the could not be identified.

I also tried to extract DNA from the final 22 samples, but the blood would not lyse so it would have difficult to aliquot. This meant that I am having to wait for a streptokinase enzyme which can lyse the blood so that DNA extraction can be done on these samples.

Finally this week I had to re test all of the samples which had shown positive results to make sure that we had not received false positives for them and we would not be wasting money sending them off for sequencing. I have had 13 positives so far so retested the DNA with PCR and gel electrophoresis. Out of the 13 positives I had, 10 of them showed positive for the second time so were sent off for sequencing I am waiting for the results to see if we can confirm the presence of any Bartonella species.

I have also been helping other people this week by running various PCR and gels in my spare time.

Our adventures

This week we wanted something to remind us of home so we decided to cook a roast dinner with all the trimmings, roast chicken, roast potatoes and vegetables. This took slightly longer than we had anticipated as we didn’t realise the oven temperatures were in Fahrenheit so had the oven on too low for a while!!

At the weekend we decided to head down to the buck 50 movie theatre, where you can watch movies for only $1.50!! They are slightly older movies that may have come out a couple of months ago but definitely worth waiting to only pay that price! We went to see Kung Fu Panda 2 and were both laughing throughout the film!! We had also gone out to dinner to a place called Mitch’s tavern which is so cheap. We ended up getting dinner and a movie for around $10 which would never happen at home.

We decided to make the most out of the lovely weather and went to the pool on Saturday. In the evening we went to see Back to the Future at the art museum open air cinema. So we sat under the stars to watch the movie which was a really nice experience and the atmosphere was brilliant. Sunday again we made the most of the fantastic weather and had a BBQ at my house.

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12
Sep
2011

This week has again been much of the same work. We continue to attempt to perfect the Western Blot, which is proving increasingly difficult as the variables that we can change are now becoming limited. We have been getting results, however these are few and far between and do not appear to be reproducible, so more work needs to be done. Even the positive control is proving to be difficult to obtain results from.

On Wednesday and Friday I continued with my cell counts which are getting there and should be finished in the next week which will be good, then we have to analyse that data, which will be tricky as there will be over 700 bits of data to organise.

On Thursday I carried out another protein assay in preparation for more Western Blots next week which was pretty straight forward as I’ve done a few in the lab before, however when you have to do the assay on 40 samples, it does become a little complicated. Also on Thursday I went downstairs to learn about the work that another lab does with Zebra fish. One of the people I work with in the lab wants to do future work with these fish because they have a similar GI tract anatomy as humans in that the GI tract is in different sections. Zebra fish are also transparent which makes it possible to use fluorescent markers to mark the tract, in addition, they have rapid growth and are easy to genetically modify in large numbers. Aside from the GI work that can be carried out on these fish, Zebra fish also are one of the few animals with heart cells that can regenerate, and therefore they are becoming essential in research to cure heart disease.

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12
Sep
2011

This week has been much of the same work as last week.  However, we needed to perfect the ChAT protein Western Blot, which was previously unsuccessful.  There are a number of variables that we can change in order to optimise the assay, for example the concentrations of the antibodies. Monday and Tuesday were spent performing another Western Blot; I have been left alone to do them, with someone just around the corner in case I need help!!!

In addition to the Western Blots, I have also helped with some mouse post-mortems.  Tissues from the post-mortems were then tested using RT-PCR. The RT PCR uses mRNA to produce cDNA and then DNA with the use of primers.  The RT-PCR is used to indicate which genes are expressed under different conditions. If ordinary PCR were to be used, it would just produce DNA which would indicate the presence of genes in the genome and not their expression.

Other than that, I have been continuing with the mast cell counts, which are nearly finished, finally!

On Friday I went with someone from my lab to watch a PhD defence, which was incredibly interesting, however totally mind boggling as I didn’t understand much of what was going on! It was a great insight into how much work goes into obtaining a PhD and just how knowledgeable you have to be.

Social Activities:

It has been a pretty quiet week as far as going out goes; we didn’t do a great deal during the week and at the weekend, the weather was pretty bad. However we did go downtown to the Science Museum, which was all about the history of science in North Carolina which was fascinating. The exhibits were great and the attention to detail was unlike anything you would see in the UK. Afterwards we went for some lunch in a little cafe and then my housemate Kristen picked us up and gave as I tour of downtown Raleigh in the car. In the afternoon we went to the cinema on campus with a few people from my lab to see ‘Crazy Stupid Love’ which doesn’t come out in the UK for a few months… amazing film!  We then found a frozen yoghurt shop, which is the healthy alternative to ice cream, however they have massive pots, which of course have to be filled  and a whole selection of unhealthy toppings which we piled on!! On Sunday we went back to the mall to go to the Cheesecake Factory again, this time we decided we would not eat a big lunch so there would be room for cheesecake, so we order a starter of nachos which were massive, and then we got cheesecake!

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24
Aug
2011

This week I have repeated another protein assay with different samples collected from pig intestines. The assay has shown that the optimum dilution of the samples is 1:100, which also fits perfectly into the standard curves that have been created. Additional Western Blots will be performed over the next few weeks. Continue reading »

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23
Aug
2011

This week in the lab I continued with my project once again. I started the week the same as last week extracting DNA from a further 48 blood samples using the robot again.  I then continued with the PCR on 16 samples and I managed to obtain three positives from these samples, which have were then sent off for DNA sequencing. I received the results back from the previous positive PCR samples and they did not contain any Bartonella DNA, but were positive for human DNA.

To try to improve the PCR’s I changed the brand of polymerase enzyme to one called Takera. I used the first 26 samples and re did the PCR and gel electrophoresis with the new enzyme. This then meant that the positive results came out as negative, so showed that there had indeed been human DNA in the samples and not the Bartonella DNA I was looking for. This week I also helped some of the other members of the lab with running gel electrophoresis on their projects.

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17
Aug
2011

This week in the lab I carried on with my project testing dog blood DNA from Canada. I extracted DNA from the next 48 samples using the robot which automatically extracts the DNA from the blood samples. All you have to do is place the blood samples, reagents and tips into a container and then the robot automatically extracts the DNA. I then quantified the DNA using the nanodrop instrument. I then went on to perform PCR’s reactions on the samples and visualised the PCR amplicons using gel electrophoresis.  However, out of 48 samples only one was positive.  The positive sample was been sent for DNA sequencing. Continue reading »

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10
Aug
2011

On Monday, I was assigned to the project I will be working on for my time in the lab. I am going to be testing blood samples from dogs in Canada for Bartonella. I had been giving hundreds of samples which all need testing, so it should keep me busy for a while!!! I will have to extract DNA from the samples and then run PCR’s on the samples. Continue reading »

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10
Aug
2011

This week has been very busy in the lab. I have been able to use some of the skills that I have learnt in the Biochemistry practicals at University of Surrey, so this was really good. The beginning and the end of the week were spent cutting tissues and staining the tissues in order to perform mast cell counts. I have made about 150 slides already! Continue reading »

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