Emily Beere

Lucy

Monday

In the lab: We discussed a lot about what to do with making more vector because we’d really been struggling to get any descent amplifications of it from the PCR. So we looked at everything we had and decided to start from the very beginning with the circular plasmid rather than trying to make more of the linear vector directly from some other linear vector reserves (because we weren’t sure exactly what our linear vector was ie was it suitable for PCR or not).

We set up a PCR of the circular plasmid then when that had finished and we’d checked it’d worked by gel electrophoresis we went on to do transformation. We put the circular plasmid into e.coli cells and grew them up on selective media over night. We also did homologous recombination and plated up one of our genes which we new was correct and would work (gene X) to use as a positive control.

Tuesday

In the lab: Adam came up with a new idea for trying to get gene V amplyfied by PCR. We made serial dilutions of the forward and reverse primes and used lots of different combinations of them in the PCR mixes to see if we could get a stronger band of our desired gene and less of the little dimers which we kept getting loads of and which were getting in the way.

We did the PCR and ran it on a gel to see the results. We had a major breakthrough; the results were really interesting and clearly showed that it was the forward primer which was mostly resposible for the dimers. In the mixes where there was less forward dimer to reverse primer we had really strong bands of our gene and a lot less dimers. This was very exciting as it was the best result we’d had so far for gene V! The plates of our plasmids, which we’d left to grow overnight had loads of colonies on them which was great so we picked a few of these and grew them in liquid media overnight for digetion the next day.

Wednesday

In the lab: We pelleted and restriction digested our plasmid which had grown in liquid media overnight to see if it was infact the right plasmid. The gel electrophoresis showed that it was the correct plasmid so we went on to do a different digestion of the plasmid to make it linear so that we could use it for the homologous recombination (this incubates overnight).  We then decided to try and get a strong, clean source of our gene V. So we ran out some of the better PCR tests from the day before on a gel to do a gel isolation of all of them.

We added all of these gel isolations together so that we had a superconcentrated gel isolate of our gene. After we’d done this we ran this out on a gel to check that we’d got a nice clean source of our gene V and that we had enough of it ie. Hadn’t lost too much during the gel isolation proccess. The result looked very good so we now have a strong, clean source of gene V ready to go on to homolgous recombination! 

Thursday

In the lab: The vector should have been cut overnight making it into an linear vector for us to use for homologous recombination. We ran the vector that had been digested overnight on a gel to see if it had been cut (using an uncut vector as a control). The gel electrophoresis showed that the vector had been cut so then we did a PCR of the vector so that it had the correct end (dictated by our primers) and now we have a linear vector to use in our homologous recombination.

Friday

In the lab: We cleaned up the linear vector we had made and measured its concentration using the nanodrop spectrophotometer but we couldn’t set up the homologous recombination yet because it was the weekend the next day. Megan in my lab was planning to come in on Sunday anyway so she will said she’d set it up on Sunday so its ready for Monday instead. So an early finish today!

After the lab: Fiona came back to mine for the weekend as Katie and Liz are both away so we thought it’d be better if she stayed at mine for a bit becaseu you can walk to more places from mine. We decided to get Ben & Jerry’s on the way home. We both got a hot fudge sundae. It was really lovely, but so chocolatly I felt completely chocolated out afterwards! We then had pizza for dinner whilst we watched the London 2012 Olympic opening ceremony.

Saturday

We went to visit the North Carolina Museum of Art for the day. We walked there along footpaths and it was absolutly boiling hot but once we got into the Museum Park which surrounded the Museum there was lots of other little paths that went into a wood under some trees so that was a nice relief.

We saw half of the outside art pieces, some of which were really interesting there was a particularly intersting one called the Cloud Chamber. We got to the main museum buildings and there was loads of stuff in there and we spent ages walking around the exhibits. There was some more outside things such as a really pretty pond/ water feature with lilies and damselflies flying around it.

After we’d seen everything we walked home via a slightly different route to see the other half of the outside art. Then finally we got home; we were so hot we went inside to cool off before doing anything else. When we’d cooled down we went to The Waffle House for dinner.

Sunday

We had a lovely lie in before making our way to Bojangles Famous Chicken ‘n Biscuits. We both got The Cajun fillet Biscuit combo with fries. On the way back from Bojangles we stopped off at a supermarket to buy some goodies!!

 

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Fiona

 This week I have been collecting RNA from the Zebra fish samples. This involved a series of steps including centrifuging and washing the samples in order to isolate the RNA, and suspending it in sterile water to allow it to be used to create cDNA. As RNA degrades rapidly, everything has to be done fairly quickly, and keeping all the samples on ice. Once the RNA has been collected, a nanodrop machine can be used to determine the concentration of RNA in the samples. About half the samples showed a good concentration of RNA, but in some of the samples the concentration of RNA was very low.

This could be caused by a variety of different reasons, such as not using enough of the Zebrafish samples, not homogenising the samples enough before RNA was extracted, or an error in the method of RNA extraction. To confirm if there was enough RNA in these samples to create cDNA, gel electrophoresis was run. This did not show any RNA present, and therefore these samples were not suitable. This means that next week I will be repeating these steps to hopefully collect enough RNA from the samples.

I was also able to continue running the PCR samples from the weeks before. So far the results have not shown anything very noticeable, but when all the samples have been run, I will be able to compare all of these results to see if there are any trends which are similar every sample.

As Katie, who I am staying with, is away at the moment, at the weekend I got the bus to downtownRaleigh on Saturday. We spent the day looking round theNorth Carolina natural sciences museum, the history museum, and of course the chocolate factory! Then on Sunday we went out in the morning for a traditional breakfast of pancakes, before coming back for another day by the pool.

 

Lucy

Monday

In the lab: I took the tick that I had found in my bedroom into the lab and everyone was very interested in it and amazed at how small it was (it was decided that it was a nymph stage).

We had another lab meeting today; the meeting was the same format as last time and was good to do a roundup of the project so far. I am now also gaining some nick names such as LT, Dr Tilsley and The silent science ninja (because I sit there quietly taking everything in).

After the Lab meeting we obtained the DNA sequencing results from the genes we’d sent off for screening. Henry showed me how to use a computer program to read the result and to manually fix anything the computer couldn’t figure out and to compare the forward reading of the gene with the reverse reading of the DNA to get the continuous DNA sequence.

After we’d looked at and fiddled with the three sequenced genes we were able to confirm that we had got the correct sequences (and in frame). So gene X, Y and Z were definitely good to go on to the next stage. We didn’t have enough plasmid vector in stock to go on to do the next lot of homologous recombination for genes V or W so we set up a PCR to amplify and build up a stock of our vector first instead.

Tuesday

In the lab: We ran a gel for the PCRs we’d done the day before, but unfortunately, it showed that our PCR had not worked. We went and looked at a paper which had details on how to amplify this vector by PCR and discovered that we needed to have used more vector (for copying) than we had done and that some of the programming of temperatures and times in the thermo cycler were slightly different.

So we set up the PCR of this vector again with these changes. Now we definitely had the right genes for genes X, Y and Z we decided to go ahead onto the next stage with them which was to express the genes (as proteins) by IVTT (in vitro transcription translation). So we mixed the genes in with some E. coli cells, some amino acids and some buffers and incubated them for 6 hours. We used GFP (green florescent protein) as a positive control.

I then went off with another scientist in our lab to identify the tick I’d brought in the previous day. We looked at it under a dissection microscope looking for any patterns on it, the size and shape of its mouthparts (palpi), the festoons on its back end and at its anal groove. It was decided that the tick was either a Brown Dog Tick (Rhipicephalus sanguineus) or a Deer tick (ixodes scapularis). I think in the end it was decided it was a Deer tick nymph.

The Deer Tick is slightly less common than the Brown Dog Tick, will feed on humans and carries disease which humans can get such as Lyme’s disease (luckily Lyme’s is not common in this area). Whereas the Brown Dog tick only carries dog diseases and the USA subtype will only feed on dogs (even though the same species in Europe is a very promiscuous feeder). The Brown Dog tick is also the only tick that can breed and build up colonies inside houses.

We all went out for lunch again: We went to The Q Shack in Northhill Mall which served the classic southern food includingNorth Carolinasfamous BBQ (pulled pork shoulder) which is what I had with sides of fried okra, mac and cheese and hush puppies.

Back in the lab: We ran a gel for the PCR we’d just done and this time it looked like we’d been successful in amplifying the vector so now all we had to do was clean it up. We also measured the amount of gel isolate of gene V and W we had (which we were hoping to do the homologous recombination with) but unfortunately there wasn’t enough so we had to go back to the drawing board for gene V and W (again!). The IVTT was still incubating so we decided to come back for the results tomorrow.

Wednesday

In the lab: We got the results of our IVTT. The GFP (green florescent protein) positive control glowed green under UV light which meant that we had achieved expression of that protein so in theory if our other proteins should have been expressed as well. So to check this we run a Western blot. These are very time consuming and have lots of little steps; we started setting ours up at about half nine in the morning and we didn’t finish it until about four in the afternoon!

We were very excited when we finally finished it and got an image from it though because it showed that all three of our proteins (from genes X, Y and Z) had been expressed! We also set up another PCR to try and get gene V and W, using different combinations of starting DNA using genomic DNA and gel isolate from previous attempts. The results of the PCR looked pretty good on the gel. Gene W was good and strong and gene V was there a bit at least! We decided to take some gel isolates of them, clean them up and then run some gels to see if they’re any cleaner and stronger.

After the lab: Fiona and I met up and went toCameronVillagefor dinner to talk about and arrange some of our future plans inAmerica. We went to The Village Draft House which is like pub really. I had the Carolina Chiliburger which was a massive burger with chilli con carne in there as well as the burger! It was very nice. Fiona had some kind of breaded chicken and it was huge!

Thursday

In the lab: We decided to optimise our Western blot to see if we could further confirm our proteins. We stripped it (cleaned it of all the things we’d put on it the day before) and then used an anti HA antibody and HRP goat anti mouse antibody) to find the HA-tags in the protein (for the initial pictures we used the His-tag markers using anti His antibody and HRP goat anti mouse antibody). The resulting image was very weak and didn’t really show anything useful so we just ignored it.

We also ran some more gels from the cleaned up DNA gel isolates and PCR products from the day before. The first gel for gene W looked good, but gene V looked really strange, we think we overfilled the well on the gel for that one so it had spilled out everywhere. So we reran them and this time they were both good although gene V was still very weak. We’re going to go on to homologous recombination with both of them but we’re not very hopeful about gene V!

Friday

In the lab: We couldn’t do a lot in the lab today because we were unable to do anything with genes X, Y and Z till we’d got genes V and W to the same stage. However, even though genes V and W were ready to go on to homologous recombination we couldn’t do them either because we still hadn’t managed to make enough plasmid vector for them. So we set up a PCR to make some more of the vector. However the results of our PCR on the gel were not good so we still didn’t have enough plasmid vector to do anything with.

Saturday

Fiona and I caught buses to Downtown Raleigh to spend the day there. First we went to the North Carolina Museum of Natural Sciences which was really good. There was a room full of massive whale skeletons which I absolutely loved! Then just lots of animally stuff (including some live animals like fish and turtles and reptiles) which was really interesting. I found out the little red birds I’d seen around were actuallyNorth Carolina’s State birds; the cardinal. Then there was another section that was full of dinosaur skeletons. It was all very good though and the exhibits were put together really well.

After the museum we went to have lunch at The Pit, which is a restaurant which serves classicNorth Carolina/southern food include the North Carolina BBQ pulled pork which is what I had. It was very busy when we got there so we were told we’d have to wait half an hour for a table or we could sit outside. So we opted for the sitting outside, it was scorching and there was no shade at all but we powered through and ate all our food just before a storm came in!

We then visited Videri Chocolate Factory which was just across the road from The Pit. We looked around the factory to see how the chocolate was made then went back to the front to get some samples. They had four flavours Dark milk (50% chocolate and the only chocolate with dairy in it), Dark (70%), Sea salt 60% dark chocolate and Pink peppercorn 60% dark chocolate.

We still had a lot of time before we had to catch our buses home so we decided to visit the North Carolina Museum of History. This was pretty good and had some interesting stuff on the history of North Carolina and on the North Carolina’s state dog; the Plot Hound.

Sunday

I got up early and helped Liz out at the stables again, after which we collected Fiona to go for a breakfast/lunch at Kristie’s Restaurant. I had two massive pancakes topped with blueberries, pecans, chocolate chips and syrup.  They were very good and I was stuffed for the rest of the day! After the breakfast/lunch we went back to Fiona’s place to go to the pool. It was a nice comfortable temperature this time and I’ve actually started to get some proper tan lines!

 

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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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