Protocol 1. Label one microcentrituge ThINQ! group\'s name. Place tube +pGLO and

Question

Protocol 1. Label one microcentrituge ThINQ! group's name. Place tube +pGLO and another-pGLO. Label both tubes with your and Exercises them in the foam tube rack. resourees to eamne the bottle of pOLO plesmd do you see? Note your obsenatios 2. Open the tubes and use a sterile DPTP to transler 150 mM Cacy) into each tube 250 ul of transformation solution a separahe plece f paper lst a that can be desarbed. For eampe 3. Place the tubes on ice. Color of colaries Size ot 1) the largst colony 2) the smalest colony 3) the majprity of colonies . Shape f colonies both 2-D and 3-D Appearance of the colonies under reguar and UV light Why do you sod the CaQ 4. Use a sterile loop to pick 2-4 large colonies of bacteria from the starter plate. Select colonies that are "fat" (1-2 mm in diameter). It is important to take individual colonies not a swab of bacteria from the dense portion of the plate), since the bacteria must be actively growing to achieve high transformation efficiency. Pick up the +pGLO tube and immerse the loop into the transformation solution in the tube. Spin the loop between your index finger and thumb until the colonies are dispersed in the transformation solution (there are no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the-pGLO tube. Why do you place the tubes on

Explanation / Answer

Ans 1- The original traits of E.coli is that it is able to grow on LB agar but unable to grow on LB+amp plates also it is unable to use arabinose and did not glow under UV light.

But these traits get altered after transformation. When E.coli is placed in transformation medium along with pGLO plasmid, genes present on the plasmid (amp, ara and GFP) get inserted into E.coli genome after successful transformation.

Now due to this reason E.coli becomes resistant to ampicillin and able to switch on the arabinose gene hence now they can grow on plates containing amp and can glow under UV light showing fluoroscent green color.

Ans 2- If the bacteria is able to grow in presence of ampicillin, it can glow in the presence of UV light if arabinose is added in the medium. Because in the presence of arabinose, ara C gene becomes active and hence GFP gene which is under control of arabinose promoter also becomes active. This results in green glow of cells.

Ans 3- when we analyse the results it shows that -pGLO plates in which plasmid is not added E.coli cells are able to grow on LB agar but are normally not resistant to antibiotic ampicillin.

After performing the procedure for mixing the E.coli cells with pGLO plasmid and giving them heat shock, the cells develops the ability to grow in presence of penicillin as well as to glow in presence of arabinose because colonies are observed on both LB+amp plates as well as on LB+amp+arabinose containing plates.

These results clearly shows that transformation has occured due to the procedure that we performed.

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