Lucy

Monday in the lab: I was told when I arrived that we’d be having a lab meeting tomorrow morning and that they wanted me to give a short presentation on what I’d been doing so far. We then reran some more plasmid minipreps and performed restriction digestions of genes Y and Z to see if we had any plasmids harbouring the correct inserts (clones), unfortunately non of the preps contained the correct insert. So we reran the assay with the last of the colonies grown on the selective plates and this time one of the preps contained the right insert! So that was genes X, Y and Z done. Just need to go back and clone the V and W genes now.

Tuesday in the lab: I went in early on this day to see some summer students present their research. It was interesting to learn about all the other research that goes on in the labs and the other techniques that I could be using later on.  Then we went back to the lab and waited around for our lab meeting at eleven o’clock. I was very nervous about presenting in the meeting, but it went really well. Adam (in charge of our lab) kept on asking me lots of questions and looking at my gel electrophoresis pictures and asking me to explain them. He also asked me a lot of general science and lab questions throughout the meeting as well, testing how much I knew and had taken in, which was good.

After the lab meeting: Henry, Megan and I went out for lunch to a Mexican called the Chile Bomba. Back in the lab, after talking things over in the lab meeting we decided to go back to try and clone the V and W again. So we went back to the beginning again to do a rerun of homologous recombination. We set up the PCR again as we did the first time, but this time with more insert (genes) than the first time (to hopefully increase our chances this time). After the PCR we cleaned them up, reran homologous recombination then plated them onto selective agar plates and incubated overnight.

Wednesday in the lab: The bacteria harbouring the plasmid with Gene W had not grown at all on the selective plates so that had failed, so we need to go back to the beginning again with that one. However, the bacteria harbouring the plasmid with the V gene had grown, so we picked those colonies off and grew them up in liquid media overnight, performed plamid preps and digesting the next day. We then made some bacterial stocks of genes X, Y and Z and decided to tackle gene W tomorrow.

Thursday in the lab: We started from the very beginning again with gene W (and gene V just in case); with PCR from the genomic DNA, this time with a gradient of annealing temperatures to try to get more of it. We also pelleted, miniprepped and restriction digested gene V from the liquid cultures. Gel electrophoreses of the restriction digestion showed that we did not have the right clone so it was a good job we’d don the extra PCR with the gel isolate in for gene V as well.

However, gel electrophoresis of our PCR showed that the bands (amount of DNA) we had was to weak/little to progress to homologous recombination. We decided to call it a day at this and try again tomorrow.

Friday in the lab: So we tried PCR again with gene V and W, but this time we also spiked one with some gel isolate from the previous gel the day before to try and increase our yield. Gel electrophoresis showed that our gene V had completely failed with barely anything showing up so we needed to try to get that gene again but our gene W had worked ok so that could go on for homologous recombination. We decided to tackle this next week. We also sent of our successful gene X, Y and Z off for sequencing.

On Saturday we went to Crabtree mall for the day. We spent the morning looking round all the shops but the main reason for our visit to this particular mall was to go to The Cheesecake Factory. I have never seen such a variety of cheesecake in my life! There were three pages of the menu devoted to just cheesecakes. It was so hard to choose which one to have but, in the end we both went for white chocolate and raspberry truffle cheesecake.

Sunday was a beach day! We got up early and headed for the beach which was about a 2 ½ hour drive away. We got to the beach and it was lovely and hot so we set out the towels, plastered on the sun cream and lay there in the sun with some homemade sangrias.

I spent quite a lot of time swimming in the sea; the water was lovely and warm and it was good to swim in once you got past the breaking waves

After the beach we went to get some food and went to a place called The Crab Pot which was nearby. It served lots of different kinds of shellfish and sides (mostly fried). I had a crab cake but got to try lots of other stuff – shrimp, fried green tomato, fried zucchini (courgette), fried pickle, fried mac and cheese and snow crab legs.

 

Fiona

This week has been fairly busy in the lab, with starting to use the Real Time PCR on some of the samples from the Zebrafish tissue. I am using a TaqMan probe in the PCR reactions to determine the level of gene expression in the samples. This gene probe also contains a fluorescent marker, and binds to the gene of interest. As the DNA is copied during the PCR cycle, the fluorophore become detached from the gene probe, causing fluorescence to be emitted. Therefore, samples with higher levels of the gene will produce more fluorescence, and computer software is used to analyse the amount of fluorescence produced from each sample.

As well as using a gene probe designed to test for the gene we are interested in, the samples are also tested for levels of expression of a housekeeping gene. This is a gene which is always expressed in every cell type, and therefore can be used as a comparison for the expression of the other gene.

Running the PCR produces lots of raw data which then has to be analysed in a spreadsheet, so this was my next job after each PCR was run.

Unfortunately, the housekeeping gene I was using ran out before I had finished all the samples, and as it takes a few days for more to come in, for the last part of the week I started planning the next experiment I would be involved with. For this project, I will be extracting RNA from Zebrafish samples and using it to create cDNA. This can then be used for PCR, similar to what I have already been doing. As samples need to be taken at specific time points, it has to be planned carefully to make sure everything is ready for each step.

At the weekend we were given a lift up to Crabtree Mall, which is a massive shopping centre in Raleigh. We had been told the Cheesecake Factory was worth visiting, but had also been warned about their massive portion sizes! It definitely lived up to its reputation, but we managed to choose from the list of just about any cheesecake flavour possible, after sharing an enormous plate of nachos. On Sunday we went on a trip to the beach, and got a chance to enjoy the hot weather before going to a local seafood restaurant.

 

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Fiona

We arrived on Monday and had a tour of the vet college at NCSU,(which looks amazing!) before settling into our accommodation. The next day we went into our labs and found out more about the projects we will be doing while we are here.

My project is helping to look at how a particular gene is involved in the immune function of zebra fish.  This involves using Real Time PCR and gel electrophoresis to investigate the levels of expression of the gene. 

On Tuesday I was shown how to carry out the Real Time PCR and gel electrophoresis, I also learned about the importance of pipetting the samples accurately and ensuring that there is no contamination in any of the reactions to be certain the results are reliable.

Wednesday was Independence Day so we had the day off to explore Raleighand visit the celebrations downtown. Unfortunately we got caught in a thunderstorm, but we managed not to get too wet and it was dry in time for the firework display in the evening! I spent the rest of the week performing more Real Time PCR and gel electrophoresis to acquire more practice before using these techniques to start on the actual project next week.

On Saturday we visited one of the local shopping malls, before going out to a restaurant in the evening. Sunday was incredibly hot, with temperatures getting up to 41°c, so just right for a lazy day by the pool near where I’m staying!

Lucy

We departed from Heathrow on Monday and arrived inRaleigh in the afternoon. We were picked up and taken to the NSCU vet school where we were given a short tour and of theVetSchool and introduced to the people we were staying with and the people in or labs who we’d be working with. We then went off with our respective housemates and did some grocery shopping before going home to bed.

Tuesday was the first day in the lab: First I was given a talk on what the project I was going to be working on; what it was about and what I’d be doing for it in the next 9 weeks. Then we went into the labs to do some PCR on some genes (V, W, X, Y and Z) from the organism in our project. After the PCR I learnt how to prepare, perform and image an electrophoresis gel in order to check that we’d successfully amplified the genes using the PCR technique. Then I was shown how to do homologous transformation to make clones of the genes we’d amplified. These were plated onto selective media and then placed in an incubator and left to grow overnight.

After the lab: Fiona and I went out with Katie (the person Fiona is staying with) for a Mexican meal which was really good.

We had a whole day off work on Wednesday because it was Independence Day! So after a nice lie in, Fiona and I met up and Liz (the person who I’m staying with) dropped us into downtown Raleigh in the afternoon for all the festivities. The streets were packed with stalls selling food and stuff, there was wrestling, melon seed spitting competitions, eating competitions and much more.

We wanted to stay for the firework at half nine though so we walked through the stalls again up to the firework end of the street. When we got to the end of the street there was a circus act performing so we watched that, then there were bands playing which we listened to then the circus act came on again with their full show just before the fireworks. We then watched the fireworks, which were pretty good.  

After the fireworks the streets were completely packed with everyone trying to get back up the street and out so we were slowly making our way up the street and we happened to walk up to and then stop by an American film crew so we ended up on TV! Not bad after just three days in the country!

In the lab on Thursday: We checked our selective plates from the homologous recombination for growth. Three (X, Y and Z) out of our five had colonies on them. We reran the PCR and gel electrophoresis for the two (v and W) that didn’t work, but unfortunately it failed again (we could tell by the gel electrophoresis this time). So then we took a few of the colonies from each of the plates that did work (X, Y and Z) and cultured them in liquid media for using in restriction digestion (the next day).

Friday in the lab: We took the liquid media cultures that had been growing overnight and pelleted the cells and then performed mini-preps to extract the plasmid DNA.  We then performed restriction digests using endonuclease restriction enzymes to cleave the inserts and see if it was the gene of interest that had indeed been  cloned.

After the restriction digestion we ran a gel electrophoresis to determine if we’d had the right insert. One of them (X) had the right insert, but the other two (Y and Z) didn’t. At this point Henry (the person working with and helping me in the lab) had to go home so I was left to pellet and mini prep some re runs for the two (Y and Z) failed tests completely by myself and other than I slight malfunction with a pipette it all worked!

On Saturday I had another nice lie in then met up with Fiona and we went to Cary Town Mall for a bit off shopping. We looked around some of the bigger shopping centres then we all went out to dinner inChapel Hill at a place called The Restaurant on the Hill because Liz’s sister had come to visit for the weekend. We both had Mac and cheese withNorth Carolina slow cooked pork which was really good and the first time I’d ever had Mac and cheese!

On Sunday I got up early this morning to go with Liz to the barn to see her horse and help her out with the feeding etc. After we’d got back I went over to where Fiona was staying because there’s a pool there so we spent the afternoon swimming in and by the pool which was lovely especially as the temperature reached 106 ᵒF (41 ᵒC). Liz picked me up from there and her sister was still here so we all went to Fresh Berry which is a frozen yoghurt place. It was really good and just what you needed on a hot day.

 

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