Question

When researchers are working with the pCRII-TOPO they will often excise the fragment using EcoRI if possible. Why?

Answer 1

The pCRII TOPO cloning is used for cloning PCR products. The plasmid has a Topoisomerase attached to the T of the insertion site.

The pCRII TOPO has a multiple cloning site that has 16 restriction enzymes: HindIII, Kpn I, Sac I, BamH1, SpeI, BsxtX1, EcoR1, EcoRV, Not1, XhoI, Nsi1, Xba1 , Apa1. Both EcoR1 and BstX1 sites are represented twice. If there is only one restriction site present, two enzymes are required for excision of insert. These two enzymes may not be compatible.

BstX1 cleaves to form sticky ends. However, the sticky ends in insert and cloning vector should be strictly compatible. Hence, this enzyme is not preferred. Further, these sites are away, albeit on both sites of the insertion site of the PCR product. This will add vector sequences to the PCR product upon excision.

There are also two restriction sites for ECOR1 in the plasmid. These sites flank the insertion sites of the PCR product. Cleavage of these sites by the enzyme cause easy excision of the PCR product without adding much vector sequences to the PCR product. Hence, this enzyme is preferred for excision of the PCR product.