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Experiment 5: Transformation and Mini-Prep of plasmids
Tuesday we assembled the Transformation reactions for plasmids PSB1A3 (our primary) and PSB1A7 (positive control). We had 3 tests; 1A3 with E.Coli, 1A7 with E.Coli, and E.Coli with water. We heat shock transformed the plasmids into the E.Coli cells, then incubated the transformed cells on plates (with appropriate antibiotics) overnight at 37°C. We also plated PSB2K3, by streak inoculation since it was already transformed into E.Coli, and also plated E.Coli cells that were not transformed.
Plates: E.Coli cells on LBA+amp and on LBA. This was to know if our competent cells are viable and non-mutated. E.Coli+water on LBA+amp and LBA. This was to check that our heat shock did not kill our cells, nor mutate our cells. PSB1A7 (positive control) on LBA+amp. This was to make sure we performed transformation correctly. PSB1A3 on LBA+amp. This was to see if we could transform our primary plasmid. PSB2K3 on LBA+kan. This was to grow more 2K3 plasmid (our secondary plasmid). The plates grew as expected, where plasmid cells grew on respective antibiotic plates, while none of the E.Coli grew on their own. Also, the E.Coli grew very well on non-treated plates.
Wednesday we picked colonies (1 colony per tube) off of our plasmid plates (1A3, 1A7, 2K3) and made 5ml culture tubes with respective antibiotic. They incubated 37°C overnight, shaking at 220rpm. 2 1A7 tubes, 4 1A3 tubes, 4 2K3 tubes.
Thursday we performed GeneJET Plasmid Mini-Prep on all culture tubes using 4 of the 5 ml. We now have 70μL of each tubes plasmid. This is to be tested next week for the correct parts. We also made 16% Glycerol stocks of what was left over in the culture tubes.