superantigen genes.help.alternative method to Southern hybridisation sought

Frans and Magda van Gils muka at muka.co.nz
Sat Feb 8 04:13:29 EST 1997


Dominiek Baetens <muka at muka.co.nz> wrote:

Superantigens are thought to be involved in generating sepsis.

I have been looking at the prevalence of staphylococcal superantigen 
genes in clinical samples of S. aureus using probes made from cloned superantigen genomic fragments.
These were excised from their plasmid vectors and purified by low melting
point gel electrophoresis and hot phenol/glass milk extraction prior to labelling.
Both DIG (digoxigenin incorporation followed by immunodetection) and radioactive labelling
were used.

Problems persisted with backgound, lack of probe specificity (despite extended purification) and 
lack of reproducibility of results -changed patterns of hybn, failure to hybridize.

Ideas? what went wrong? alternative techniques?
I now have one week left to write up this project as part of a Masters of Medical Science dissertation.
help.

Dominiek Baetens

> 




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