Towards the end of the placement

It's been almost a week since I last wrote and now my placement is heading towards its finish next week.

This week I finalised cloning the plasmid with mutated HDAC5 gene that I ligated onto pCDNA backbone. I got around 15 colonies of bacteria that grew on agar plates with ampicillin, so I picked 10 of them and extracted DNA using MiniPrep kit. Then I had to check if what I isolated was exactly the plasmid with the mutant gene, so I digested it with a restriction enzyme together with wild-type plasmid to look for an additional 0.7kb piece of DNA, specific to mutant-carrying plasmids. I got this picture:

which shows that 4 of my extracted plasmids (to the left) produce a small piece after the restriction, which is absent in wild type (second right). On the right there is DNA Ladder.

I also carried out my last transfections on Tuesday, this time using a new delivery system, FuGENE, which turns out to really enhance the transfection efficiency. I'm going to stimulate the fibroblasts tomorrow and do my last live imaging on Monday. Now I'm in charge of another live imaging, transfected a week ago by Sangeeta using FuGENE and it looks really promising.

I also quantified another staining of Becky's experiment and it is a bit confusing.

To finish with an extra piece of experience, today I've started my first ever Western Blot, using old Sangeeta's neuronal extracts to test phospho-HDAC antibody. I've started a polyacrylamide gel, I prepared a transfer buffer (on my own) and I'm waiting before I start blotting.

Meanwhile I filled up MiliQ water container and took some tubes and bottles for autoclaving. It will be a bit sad to leave the lab where I feel well settled in.

Enough for now, I'm going to take another picture of my live imaging!

The best week so far

The fifth week of my placement has just finished. It was really a good week.

My immunocytochemistry of Becky's experiment revealed some very interesting results. For the first time I also quantified my results (I learned how important it was during Cellular & Molecular Imaging module last term) by counting and grouping cells from the images I'd taken. Sangeeta thinks she might publish these results, so maybe I could even get into a publication!

Second, we finally managed to get nice images from live imaging, without microscope breaking or excessive cell death.

When it comes to laboratory practice, I also created a new plasmid with mutant HDAC protein and transfected bacteria with it. And I have a few colonies! I will isolate the plasmid next week and hopefully transfect some fibroblasts with it.

This week was very rewarding. I brought some of the experiments until the end. I didn't start many new ones, but having already 3 experiments in the incubator is really a lot! I keep passaging fibroblasts in my flask so that they're ready for new transfections next week.

Promising results - finally?

I haven't written since Thursday, so I'll try to summarise the last few days.

On Friday I had quite a lot to do, but managed to efficiently organise my work. The observations of last immunocytochemistry of Becky's time lapse experiment brought promising results, so we decided to repeat the staining and stain nuclei with Hoechst dye after the weekend. I also stimulated my fibroblasts and fixed them for staining on Monday. I changed media of my other experimental cells and passaged the flask with my fibroblasts. At the end I cut the piece of DNA out of the agarose gel, so that I could extract it later and create mutants.

On Monday I completed the Becky's immunostaing, treated my stimulated fibroblasts with primary antibodies and set up live imaging (again). I also changed media, passaged cells and did quite a few "housekeeping" tasks, especially to do with shuttling between the lab and autoclave room. I like it, though. It makes me feel responsible for the laboratory I'm working in.

Today seems to finally have brought some nice images. My overnight live time course imaging looks interesting, but it will need a thorough analysis before drawing any firm conclusions. However, I also spent almost an hour looking at Becky's stained slides and they look REALLY interesting! I'm looking at the images now and putting them in order and there is a suggestion of a nice pattern.

I already finished staining my stimulated fibroblasts and put them on slides. I'll probably observe them tomorrow. If there's anything else for me to do today, it'll probably be extracting DNA from the gel. But I'm not sure yet.