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.




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.


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.


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!


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!


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.


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.


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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