Consider the following information to answer questions 1922

Consider the following information to answer questions 19-22: You designed an experiment to clone a 0.8Kb fragment digested with EcoRI and HindIII into the unique EcoRI and HindIII restriction sites of a pUC vector. A double digest of the vector generates two fragments of 7.2 Kb and 0.016 kb. In order to minimize reassembly of the vector, the 0.016Kb fragment was removed from the double digest preparation of the vector.

Transformations of the following samples were then performed:

a. 30 ng of EcoRI digested vector which was treated with DNA ligase

b. 30 ng of EcoRI + HindIII digested vector which was treated with DNA ligase

c. 30 ng of EcoRI + HindIII digested vector mixed with 20 ng of the digested DNA fragment and then treated with DNA ligase Below is indicated the number of colonies obtained for the different transformations.

Unfortunately, the plates representing the different transformations were not labelled.

Plate 1: 5 colonies of color x + 40 colonies of color y

Plate 2: 348 colonies of color x + 2 colonies of color y

Plate 3: no colonies observed 15

1. What was the ratio between insert : vector in reaction “c”?

2. Which plate has the highest probability of containing colonies representing plasmid recombinants?

3. Which plate has the highest percentage of intramolecular ligation events?

4. What is the highest number of blue colonies observed?

ANSWER ALL OFD THESE

Solution

1. The ratio between the insert: vector in reaction c is 20x7.2/ 30x0.8 =6

The ratio between the insert: vector in reaction c is 6:1

2. Plate 1: 5 colonies of color x + 40 colonies of color y has the highest probability of containing colonies representing plasmid recombinants

3. Plate 2: 348 colonies of color x + 2 colonies of color y has the highest percentage of intramolecular ligation events

4. The highest number of blue colonies observed are the self-ligated plasmid.