disruption vector

[sruswandi at eudoramail.com]

I am trying to construct a disruption vector. Initially, I subcloned the genomic DNA of my gene of interest which is about 1.6 kb.  I subcloned it into a pGEM T-easy vector(3kb).  Next, I extracted my disruptant which is an hph cassette (3 kb) from pSH75.  I used two enzymes, PvuII and EcoRV to excise the cassette.  I then linearized the pGEM T-easy vector with my insert on it using EcoRV and did SAP treatment to prevent religation.  I then did transformation to insert the hph cassette on the EcoRV  site of my gene of interest.  There were no colonies in my control plates (vector only).  I then picked up transformants and digested separately with EcoRV to linearize the plasmid.  However, instead of getting a single fragment, I got two fragments (~5 kb and ~2kb) .  The control plates indicate that there was no religation of the vector but the fragments are not the expected ones although their sum equals that of the expected.   How should I pursue construction of my disruption vector?  Thanks in advance.

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