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disruption vector
Posted by: sruswandi (IP Hidden, New member, 0)
Date: June 17, 2002 06:55AM
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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