4. You are new to the lab and you want to impress your fellow graduate students

Question

4. You are new to the lab and you want to impress your fellow graduate students so you have volunteered to make the next batch of electroporation competent E. coli cells. You follow the lab's protocol with no obvious problems and carry out a transformation to determine the transformation efficiency of your newly made cells. You use 1 ul of a plasmid stock labeled 10ng/ul in the transformation and end up plating 251 of the 1 ml transformation mix to a selection plate. After overnight growth at 37C you count 149 colonies. a. Based on these numbers, what would you estimate is the number of E. coi cells in the 1ml mixture that are likely to have taken up a plasmid? (2pts) b. What would you calculate the transformation efficiency of the competent cells that you made? Report as "cells/ug" of plasmid. (2pts) c. Based on your calculation, how confident are you in your competent cell preparation skills? Why? (1pt)

Explanation / Answer

1. We took 25ul of 1ml transformation mixture

Colonies formed in 25ul=149,

And 1ml contain=1000ul,

So, no.of E.coli cells in 1ml mixture will be=149*1000/25=5960 cells.

2. Transformation Efficiency =no. Of tranformants/ug of DNA*Final vol of recovery(ml)/Vol. Plated

DNA used for transformation = 1ul X 10ng/ul=10ng

. 1ng=1/1000ug,

. So, 10ng/1000=0.01ug.

No. Of tranformants =149

ug of DNA=0.01ug

Final vol. =1ml=1000ul

Vol.plated=25ul

Apply tha above mentioned formula,

Transformation Efficiency =149/0.01*1000/25=5.96 X 10^5 cells/ug.

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