Question

You clone in the kanR gene (confers resistance to kanamycin) at the EcoRI site in pBLU, perform transformation into E. coli DH5α cells, and plate the transformants onto the following plates: LA, LA + ampicillin, LA + ampicillin + X-gal, LA + kanamycin.

a. What is a possible explanation if no growth is observed on the LA plate?

pBLU was not digested by EcoRI fully

The kanR gene did not ligate with pBLU

E. coli DH5α cells were not viable and the kanR gene did not ligate with pBLU

E. coli DH5α cells were not viable and the water bath for the heat-shock procedure was set to 100ºC

pBLU was not digested by EcoRI fully and the water bath for the heat-shock procedure was set to 100ºC

b. What is the purpose of plating on the LA + ampicillin plate?

c. What is the importance of having ampicillin in the X-gal plate in order to perform a blue/white screen?

d. What color(s) are the colonies growing on the LA + ampicillin + X-gal plate?

All blue colonies because the lacZ gene encoded in pBLU remained intact
A mixture of blue and colorless/white colonies because the blue colonies indicate transformed cells and the colorless/white colonies indicate untransformed cells
All colorless/white colonies because the lacZ gene encoded in pBLU was disrupted by the kanR gene
A mixture of blue and white colonies because the ligation product has a mixture of self-ligated pBLU and those that ligated with the kanR gene

Answer 1

Answers-

a. What is a possible explanation if no growth is observed on the LA plate?

E. coli DH5α cells were not viable and the water bath for the heat-shock procedure was set to 100ºC. (We don't know whether ligation of kanR gene with pBLU happens or not, because if DH5α cells were not viable then they can not grow on LA plate. also if we give high temprature for heat shock then it may cause cell death). Generally heat shock temperatures between 42 and 47 degrees C used.

b. What is the purpose of plating on the LA + ampicillin plate?

The plasmid pBLU has ampicillin resistant site. So, if we plate transformants onto the LA + ampicillin plate then only the bacteria having transformed plasmid can grow. Non- transforments are unable to grow on LA + ampicillin plate.

c. What is the importance of having ampicillin in the X-gal plate in order to perform a blue/white screen?

Only bacteria having transformed plasmid would grow on ampicillin, and when we perform a blue/white screen we only need transformed colony to grow on our plate not any other contamination. So, the transformed colonies having plasmid + insert would form white colonies and transformed colonies having plasmid only would form blue colony on LA + ampicillin + X-gal plate.

d. What color(s) are the colonies growing on the LA + ampicillin + X-gal plate?

A mixture of blue and white colonies because the ligation product has a mixture of self-ligated pBLU and those that ligated with the kanR gene.

those that ligated with the kanR gene give white colonies due to disrupted lacZ (beta galactosidase) gene and self-ligated pBLU gives blue colonies due to intact lacZ gene.