Week 1– molecular Reagent Preparation

As I mentioned in the abstract, the goal of my research is to investigate mitochondrial features and its relationship with neural progenitor cells (NPCs). Therefore, it is necessary to construct certain plasmids (i.e. molecular reagents) to mark both the NPCs and mitochondria. Thus, constructing a vector plasmid requires three donor plasmids (see below): 1. a promoter region (5′ entry plasmid); 2. a sequence that is responsible for marking NPCs (middle’ entry plasmid); 3. a sequence that is responsible for marking mitochondria (3′ entry plasid). For this week, I put a sequence that generates red florescent protein into a 3′ entry plasmid as a donor plasmid.

gateway

MF_001

The sequence is obtained using PCR and its sequence is linked below. (https://drive.google.com/open?id=1Hmeyrbq0VyltdPoVP4EdJbbGu6o1Eaw_) This sequence was originally from a plasmid called 58425, which contains a Cox 8 pre-sequence followed by a TagRFP_T sequence, which makes it targets mitochondria.

I then performed two restriction enzyme digestions to create stick ends at the end of sequences (58425) and its backbone (recipient plasmid 80825), a rSAP reaction to prevent the backbone from self recombining, and a ligation reaction to combine 58425 and 80825 to generate my first donor plasmid — MF_001 (a 3′ entry plasmid containing mitoRFP sequence). It is verified by RE digestion and sequencing.

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