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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