Question

Devise a restriction analysis method to confirm the desired recombinant; use a single most appropriate enzyme. Calculate the expected sizes of the restriction fragments from each, the vector and the desired recombinant

Answer 1

Restriction analysis method for screening the recombinants requires four steps:

1. DNA extraction from the clone.

2. Restriction digestion of DNA with an appropriate enzyme.

3. Electrophoresis based separation of the DNA fragments based on their molecular-weight.

4. Comparative analysis of the sizes of the digested products with that of the expected based on the restriction map of the desired clone.

For choosing an appropriate enzyme for restriction digestion, two factors need to be considered:

1. The DNA fragments should be of the size that can be separated on the agarose gel.

2. The difference between the sizes of the separated fragments should be distinguishable.

Expected sizes for the restriction fragments for the vector and the desired fragment:

- If the given restriction enzyme has two restriction sites in the vector and insertion is done at one of those. So, after insertion only one cut will be introduced and a linear-fragment will be obtained.

Suppose Vector size- 6200bp (approximate values)

Uncut vector	Un-recombined vector	Recombined Vector
6200 bp	4000	7400 bp
	2200

So, the size of the recombined fragment is 1200 bp as identified by running the gel.

Further, confirmation can be done using another restriction enzyme specific for the recombined vector but not for the non-recombined and based on the sizes of the fragments generated, one can conclude something.