Question

explain the need for the NdeI digest prior to the second transformation of the pUC19M. how much of a purification of mutated plasmids resulted from this digest?

Answer 1

NdeI is an isolated strain of Neisseria denitrificans and is an endonuclease enzyme.

Ndel is a type II restriction enzyme that is particularly designed to cut at specific DNA sequences. In the pUC19M plasmid first time digestion is done with the random endonucleases which cleaves randomly the DNA sequences but with Ndel second time digestion allows it to cut at the specific site which allows the gene of interest to be incorporated at the target site and also allows its expression to be identified. With this enzyme the annealing temperature is also high so it requires specific conditions to maintain the incorporation it into the plasmid.

When a mutated plasmid is digested with Ndel it gives good results as it works specifically on a particular target sequence that has 5' CATATG sequence. With the help of Ndel mutation in the plasmid can be seen easily because of its property to cut at specific sites of DNA sequences.