Anabel Clements

18
Oct
2011

Again this week I have been continuing with PCR and gels for various studies.

I have been testing samples from the study previously described looking for the competency of the cat flea as a vector. In this study Bartonella henselae is used as the infectious agent and to see if the fleas have passed it from one animal to the next. This means that all of the positive results we get should be B.henselae positive. However, for a few of the samples I have tested I received positives for Bartonella koehlerae, they also can back from sequencing as B. koehlerae. This means that somewhere during the experiment contamination with B. koehlerae has occurred. If this is the case it means that all of the experimental data will have to be scraped and it may have to start again. They will have to work out how B.koehlerae has entered the experiment and on first thoughts it is believed that it may be due to the fleas which could have been infected with B. koehlerae before the experiment started.  

Our adventures

This week we did a Segway tour of Raleigh as we thought it would be the perfect way to see the sights of the city and downtown area. We took an hour tour which took us down the main street, to the governors’ mansion and to other important areas of the city. On the same day, Bugfest was going on in the city. This is a celebration of bugs and there is even a bug cafe which serves chocolate coated crickets and mealworms in sauce. Me and Jade did not try any of the things as it didn’t look like the most pleasant thing in the world. However, we did get a funnel cake, basically a giant sugary doughnut!! Sparkfest was also going on in downtown and this is a celebration of the arts, people were doing chalk drawings on the street and there was various music and dancing happening down the main street it was a really good atmosphere although trying to dodge all the people and artwork whilst on a Segway was difficult to say the least.

On Sunday we decided to go to the cheesecake factory for one last time before we left. We decided that we would just have cheesecake this time as in previous occasions we have had other food and been too full for cheesecake even if we have only shared a starter!! We even got a piece to take home for the next day so we could try as many flavours as possible!!

Posted in Uncategorized | Comments Off
18
Oct
2011

 

This week in the labs I have been continuing to test various samples using PCR and gel electrophoresis.  This includes samples from the study I have previously mentioned, where I was testing for competency of the cat flea as a vector for Bartonella Henselae. This week I have also carried out manual DNA extractions on a few pieces of tissue. This process involved lysis of the tissue using protinase K, then cycles of adding various buffers and centrifugation. One of the samples yielded a very high DNA concentration as measured by the nanodrop spectrophotometer, so I had to repeat the last step in the process in order to dilute the DNA concentration as otherwise it would be too high to complete PCR and Gel electrophoresis and the results gained would not have been accurate.

This week I have also received the DNA sequencing results back from the positive samples I had obtained from the Canadian dog blood. Unfortunately, all the sequencing reactions failed to yield sequences that matched what we were looking for. This is a shame as it would have been nice to have been got one positive from all of my samples tested. However, this could be a good thing in the long run as this may mean that Bartonella species, especially Bartonella vinsonni subspecies berkhoffii type 4 has been eliminated from this area. However, more information is required before this assumption can be substantiated, including repeating the DNA sequencing.

Posted in Uncategorized | 72 Comments
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.

Posted in Uncategorized | 2 Comments
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.

Posted in Uncategorized | Comments Off
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.

Posted in Uncategorized | 1 Comment
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.

Posted in Uncategorized | Comments Off
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 »

Posted in Uncategorized | Comments Off
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 »

Posted in Uncategorized | 5 Comments
27
Jul
2011

We arrived on Wednesday and settled into our accommodation. Jade and I are both staying with NCSU students who live a 10 minute walk from each other so we can meet up really easily.

On Thursday morning we went straight into our respective labs, mine being the Vector Borne Disease lab of Dr Ed. Breitschwerdt.  This lab has two sections diagnostics and research; the diagnostics section focuses on serology and using antibodies to diagnose diseases in cats and dogs and the research section which uses PCR and gel electrophoresis to research into vector borne pathogens such as Bartonella and Babesia. Continue reading »

Posted in Uncategorized | 1 Comment