Question

BACTERIAL TRANSFORMATION PROTOCOL AND QUESTION

1. You have a tube of pGLO plasmid at a concentration of 15 ng/uL

2. Label two microfuge tubes; one with "+ pGLO" and the other "-pGLO" Add 250ul of cold transformation solution to each tube and place the tubes on ice.

3. Transfer 10 ul of the pGLO plasmid DNA only to the "+ pGLO" tube containing transformation solution and cells. Mix the DNA solution with the cell suspension in the tube.

4. Add 250 ul of LB broth to both of the tubes.

8. Mix the tubes gently and then inoculate 200 ul onto the appropriate plates.

Plate -pGLO on the LB plate and onto the LB-Amp plate
Plate + pGLO onto the LB-Amp plate and the LB-Amp-Ara plate

After following the protocol, you count 175 CFU that glow when the plate which contains LB-amp-ara, is illuminated with UV light.

Question: What is the transformation efficiency (CFU/ug plasmid) obtained from Protocol? Show your work.

Answer 1

Answer: Transformation efficiency is calculated by using the below formula

Transformation efficiency = Total number of colonies growing on the agar plate/Amount of DNA spread on the agar plates (μg)

Given, Number of colonies = 175

Concentration of DNA is 15 ng/μl and the amount added to the +GLO tube is 10μl. So, the final concentration of DNA in the  +pGLO tube is 150 ng or 0.15 μg (1000 ng =1 μg. So, 150 ng = 0.15 μg).

Transformation efficiency = 175 / 0.15 = 1166.6 CFU/μg.