Week 4 Diary

Day 15
After the failure of the neurogenin 3 colonies, we had to first repeat the BP recombination reaction, where a gene was cut out of a plasmid and the neurogenin 3 gene inserted in its place. We then also repeated the steps using the E. coli bacteria to produce colonies containing the replicated neurogenin 3 plasmids. This involved incubating the plasmids with the bacteria, so that they would be taken up by the E. coli and then spreading this mixture onto plates of agar containing kanamycin as before, allowing time for colonies to form. Tomorrow, we will know if this has been successful the second time round: there should be many discrete colonies on the agar plate. The repetition has been a bit annoying but at least it feels like a real taste of research science, where, of course, mistakes are regularly made.
Next job of the day: make mini preps. This was also a repeated step, after the mini-preps before did not turn cloudy. Remember: we thought this was because the bacteria had died because the culture solution contained the wrong antibiotic, one which the E. coli had no resistance against. We took six samples of discrete colonies on each of the neurogenin 1 and 2 agar plates and added them to the mini-preps. Tomorrow, they should be cloudy.
Finally, we dissected some of the chick embryos to get a better view of the staining. It has produced some revelations, particularly for CNeuroM. I’m saying nothing until my project report is finished!

Day 16
Time for a change. After repeating the mini-prep step twice for the neurogenin 1 and 2, (successfully the second time!), we decided to use a slightly different method to continue the cloning of the neurogenin 3 plasmid. For 1 and 2, we made lots of mini-preps, some of which we hope will contain a pure plasmid sample, and the neurogenin plasmid that we want. This is quicker, but it means producing many mini-preps. For neurogenin 3, we used a method where we took samples of several colonies, and performed a PCR on them, in order to amplify the plasmid within. For each sample, we also transferred some bacteria onto a numbered agar grid, with the numbers corresponding to those on the PCR tubes. Therefore, once we had run the PCR and then run the product of this on a gel to see which ones contained a pure plasmid, we could go back to the grid to choose the colonies that were cloning the plasmid we want, and produce mini-preps of these (so fewer mini-preps have to be made, and we can be more confident in our mini-preps).
Back to neurogenin 1 and 2 now. The mini-preps of these had successfully produced many bacteria. Next, we had to extract and purify the plasmids inside them. A lysis buffer emulsified their membranes, thus bursting the cells and releasing the contents within. Next was a simple step by step process of DNA purification using ionisation chromatography. Then, for each now pure plasmid, we performed a restriction digest. This process basically uses an enzyme to cut out the neurogenin gene from the plasmid, ready for us to amplify the gene itself, to create templates of it.
Overall, it was a particularly confusing day, flipping from neurogenin 1 and 2 , to neurogenin 3 and back to 1 and 2 again., using tiny test tubes with clear liquids (labelled, of course!), trying to remember what you ad done with each of them so far, and what to do with them next. Still, again, this is research science in action, and you get used to multiprocessing and having the confidence in yourself that what you are doing is correct, even if you are moving 1μl of one clear liquid into another clear liquid(so barely anything comes out!) and thinking “ I haven’t done this one yet, have I!”

Day 17
The final day of the project! And the final steps to creating the neurogenin templates ready for other scientists to use in their in situ hybridisations. Their results will give data to add more genes to the timeline of neuron gene expression that I am in the process of determining.
The first of these steps: extract the plasmids from the neurogenin 3 E. coli colonies. This was just the same as for the neurogenin 1 and 2 colonies yesterday, using a lysis buffer and then DNA ionisation chromatography. The next step, just like yesterday, was to “cut-out” the neurogenin 3 gene from these plasmids. However, unlike yesterday, I used a PCR instead.
While I was waiting for this reaction to happen, I took the opportunity to take some photos of the dissected chick embryos, to produce detailed images of the staining in the brain regions. These will be very useful when analysing the roles of each of the genes, as they will give the best view of where precisely in the neural tube the genes are activated, and thus give clues as to their role.
This done, it was the final step of the project. Just as I had started with a template PCR, so too did I finish with one. Now I had copies of the three neurogenin genes, it was time to amplify these copies to produce three separate solutions containing a high concentration of DNA fragments, containing each gene. So, with the success of each of these PCRs confirmed by the running of a gel, the three templates can be sent off for sequencing to check they are in fact copies of the neurogenin genes. Once the template sequences are confirmed to be those of the neurogenin genes, the templates will then be used in another set of in situ hybridisation. Done. Or not?
My part of the project is finished, but this is scientific research and there is always more to learn. So the research continues and perhaps in the future this research may contribute to the comprehensive understanding of neuron differentiation. Then, we may be able to use this understanding to produce neuron cells from stem cells and perhaps even produce neurons to treat stroke!

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