Question

1. Why is it necessary to first transform DH5 alpha, rather than simply using BL21(DE3) to select circular plasmids from the ligation products?

Answer 1

The DH5 alpha has a recA mutation, so it does not do heterologous recombination which ensures a higher insert stability . Also, it lacks some endonucleases which might digest the plasmids during the isoation procedure.

BL21 on opposite is engineered for protein expression purposes. It is deficient in some proteases, so it will not digest recombinant proteins - or more precisely, it will do less. The often employed T7 RNA polymerase is expressed in the BL21 (DE3) strain from a genomic copy (introduced via a DE3 lysogen), upon induction by IPTG