In a paragraph please explain stepbystep what you did in the

In a paragraph please explain (step-by-step) what you did in the lab pGLO Transformation in a way that anyone could repeat the experiment

Solution

1. The first step is straking of plate with E. coli in Agar plate and incubated at 37°C, they should be used within 24–36 hr. Delay beyond 36 hr will compromise transformation.

2. Inoculate A single colony that is approximately 1 mm in diameter contains millions of bacterial cells. To increase transformation efficiency, students should select 2–4 \"fat\" colonies that are 1–1.5 mm in diameter. Selecting more than 4 colonies may decrease transformation efficiency.

3. The transfer of plasmid DNA from its stock tube to the transformation suspension after adding wait for 10 min

4. Then Heat The procedure used to increase the bacterial uptake of foreign DNA is called heat shock.The tubes containing the cell suspensions must be taken directly from ice, placed into the water bath at 42°C for 50 sec and returned immediately to the ice. The absence of the heat shock will result in a 10-fold decrease in transformants.

6. Spreading Transformants and Controls Delivering an excess of transformed culture to the plates with the disposable transfer pipet is counterproductive because the plates may not absorb the additional liquid and spreading will be uneven. Spread the suspensions evenly around the surfaceof the agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface.

7. If you plan to follow the pGLO bacterial transformation experiment with the GFP purification kit. you must save the pGLO-transformed bacteria grown on the LB/amp/ara plates. Better to store at 4 degree.