Restriction digestion of the phage display vector, pHEN

13 Restriction digestion of the phage display vector, pHEN-1 

The pHEN-1 vector (see Section 12) contains the restriction sites SJil (located 3 of the pelB leader and Notl (located 5 of the c-myc epitope tag) for directional cloning of antibody scFv genes cut with the same two enzymes (see Protocd 10). 

The pHEN-1 vector Is first digested with S I restriction enzyme followed by digestion with Notl. Finally the double digested SJil/Notl pHEN-1 vector is cut with PstI (located in the polylinker, between the S I and Notl sites) to reduce the vector background (i.e. religation of the vector on itself). 

Digest pHEN-1 with S/il by adding the following components, in the order stated, to a 1,5 ml microcentrifiige tube  

Mix gently by pipetting the reaction mixture up and down. Follow with a quick ( touch ) spin-down in a microcentrifuge.  

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