PHEN-1 vector

Purification of pHEN-1 vector by equilibrium centrifugation in CsCI ethidium bromide gradients... 

The pHEN-1 vector (see Section 12) contains the restriction sites Sfil (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 Protocol 10). 

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

Adjust the concentration of digested pHEN-1 vector to 200 ng/pl with water. 

Using a 3 1 molar ratio of restriction digested pHEN-1 vector and scFv insert," set up the following reaction in a 1.5 ml microcentrifuge tube ... 

Determine the size of the library by plating tenfold dilutions of transformed TGI cells from 10 to 10" onto 2 x TY-AMP-GLU agar plates. Assess background vector religation by ligating and transforming triple digested pHEN-1 vector in the absence of antibody scFv insert. 

RSP anneals 5 of the antibody scFv insert, just before the Hmdin site in pHEN-1 vector and as the following sequence 5 CAGGAAACAGCTATGAC 3. ... 

A PGR band of around 950 bp will be seen using the RSP and gene 3-28 primers on the pHEN-1 vector containing an insert (antibody scFv). A negative result (Le. no insert) will produce a band of 212 bp. 

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