Imagine you wanted to use a human genomic DNA library to clone the human gene for insulin. You will be using the rat insulin gene sequence as your DNA probe. In what order would you perform the following steps to accomplish this goal?

1. Use autoradiography to identify colonies containing DNA that hybridized to the probe

2. Grow transformed cells on media with antibiotics and X-gal for blue-white screening

3. Ligate genomic DNA and vector DNA

4. Cut genomic DNA and vector DNA with restriction enzymes

5. Hybridize library DNA with labeled probe for the rat insulin gene

6. Transform bacteria with recombinant plasmid

Solution

The basic steps for cloning to synthesize any protein in bacteria are as follows –

a) The first step is to cut the plasmid using a restriction enzyme and then

Pasting it into the gene using DNA ligase to make recombinant plasmid

b) This plasmid is then introduced into the bacteria to take up the foreign DNA and grown into colonies by producing identical bacterias

c) Methods such as restriction enzyme digestion and PCR are used to check for right plasmid with in the bacterial colony

d) Once the right bacterial colony is identified it is gown into large cultures to translate mRNA to human insulin protein.

e) Once the bacteria burst and releases free insulin it is extracted and purified.

Hence, the correct order is

4, 3, 6, 2, 5, 1