Question from Colombia

Edgar Andres Pulido Bravo via methods%40net.bio.net (by eapulidob At unal.edu.co)
Thu Nov 16 12:03:10 EST 2006


Drs. 

I’m addressing to you about some troubles we are having with the in situ hybridization technique. 

Currently we are working with in situ the hybridization to detect Streptococcus agalactiae in tilapia tissues. However, the technique has not been working well, neither on tissues nor on nylon membrane (in spite of using DNA purified from bacteria, as a control positive). We have tried with two probes (length fragment 860 and 190bp) labeling with biotin dCTP incorporated by PCR (on the nylon membrane the probe shows a good labeling). On the slides, with tissues fixed with formalin, we get only a very low signal and just in cerebral tissue.    

Attached you can see the protocol we are using. We would like to know if you can give us some comments or recommendations to get better results. 

Thanks a lot. 

Andres Pulido B. 
National University from Colombia


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