Lucy

Monday

In the lab: We had our weekly lab meeting to decide what to do next with our projects. We decided that the vector we’d made was good to go on to use in homologous recombination experiments. However, at some point we would need to send it off for sequencing to check it is completely right. Megan had set up a homologous recombination of our gene V (that we’d got from our new serial dilution of primers for PCR method) and gene W (which was from the normal version of the PCR). The bacteria containing the construct with gene W hadn’t grown on the selective plates so it had not worked at all and only one colony was recovered for gene V. So we picked that one colony and grew it up in selective liquid media overnight ready for digesting the next day.

Tuesday

In the lab: our bacteria containing the construct with gene V grew in the liquid media.  However, when we did a restriction digest of it, it turned out that is wasn’t the desired clone so that had failed. So we needed to try homologous recombination with it again (and with gene W). But we decided to try and get a more successful amplification of gene W first by doing our new optimized primer dilution PCR with it. So we did that and pooled together the best PCR mixes to make our gene W gene stock.

Lunchtime: Got a free lunch today at the lab! There was a laboratory company selling their products and to entice people in they were giving out free NC BBQ.

Wednesday

In the lab: We retried the homologous recombination of gene V and gene W, but this time we used fresh competent cells and used two different ratios of insert to vector (one was the one we’d already been using (3 or 4:1)and the other was a simple 1:1 ratio) to see if we got better results. So they were all plated on selective agar overnight.

Lunchtime: We went out for lunch again today. This week we went to Mitch’s Tavern which is and American pub. It was buzzing in there and amazingly busy. I got the Mexican chilli which came with cheese, salsa, sour cream and tortilla chips along with the bowl of chilli itself.

Thursday

In the lab: All of our plates had grown a fair few colonies on them! Big success! By looking at the numbers of colonies for the different ratios we’d used there didn’t seem to be any correlation there so we put our previous failures down to old competent cells. So we picked three colonies for each gene and grew them up in selective liquid media overnight.

After the lab: Fiona came back with me on the bus and we had our now weekly, Ben & Jerry’s then we went toRidgewoodshopping centre to look at some NCSU merchandise.

Friday

In the lab: All three of the colonies for each gene had grown up in the liquid media overnight. So we picked two of each of them to go on to pellet and restriction digestion. So we did the pelleting and restriction digestion. Our results showed that we’d successfully got a clone of gene V (at last!). We hadn’t got one of gene W.  However, we still had one last colony in liquid media to try first before concluding failure. So we took that last culture, pelleted it and did a restriction digestion. The results showed that we’d also successfully clones gene W as well! This was a great result as we can now move on with the project! So we made bacterial stocks of all the successful genes ready for Monday.

Saturday

We went toCrabtreeValleymall again today. To start off the day we had a pretzel for breakfast. I’d never had a pretzel before and got a cinnamon sugar one. After our pretzels we went to Alumni Hall which was the shop we’d seen the other week which sold loads of NCSU stuff.

We wanted to go to The Cheesecake Factory again but didn’t think we could fit any other courses in other than the cheesecake itself. So we went to the take out cheesecake counter in at the side of the restaurant instead of eating in. It was incredibly hard to choose but in the end Fiona chose the Oreo flavour Cheesecake and I went for the Mango and Key Lime Cheesecake. So we got our cheesecake and went to sit outside in the sun to eat them. My one was very good; I’m not entirely sure what a “key lime” is but it tasted nice and citrusy and I thoroughly enjoyed my cheesecake, definitely going to try key lime things again.

Sunday

We went toCameronVillageagain today to look around the little shops there and to get some things to eat. We were a bit hot after walking from the bus stop toCameronVillageso we decided to go to Goodberry’s to get some frozen custard first.

As with most places here inAmericathere was a huge amount of choice. In the end Fiona went for a chocolate brownie sundae and I went for the days special parfait which was vanilla custard with raspberries and blackberries layered into it, topped with cream and a cherry. Afterward we went to look around some of the little gift shop type places. First we went into Ten Thousand Villages which was a shop which sold fair trade things from all round the world. Lastly we went to Sugarland which sold cupcakes, ice cream, drinks and some other sweet pastries. 

There were loads of cupcakes, many unusual flavours as well so it was very difficult to choose. I went for crème brulee flavour one (which had a raspberry on top and in the centre) and a mango flavour one (sugar sweet on top and a bit of mango in the middle). Fiona got a lemon flavoured one and a strawberry cheesecake flavoured one. They were very messy to eat as they had a massive swirl of flavoured cream on top of them which got everywhere and fell off whilst you tried to eat it!

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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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