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!
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