Anyone aware of a protocol for inactive bovine tissues that are infected with a select agent (Brucella spp.), that can be used for downstream molecular applications (PCR) with commercial DNA extraction kits (Qiagen DNeasy)? We have tried proteinase K, but this is needed at very high concentrations to where it is cost prohibitive. We are unable to do Trizol as there doesn't appear to be a protocol that would allow Trizol treated tissues to be extracted with commercial DNA extraction kits.
Hi Simon,
We did look at this but you run the risk of denaturing the DNA with the temperature need to kill Brucella abortus.
Hi Noah
I can only speak from my own experience but I have heated bacterial preps to 120deg for 11 mins (suspended in guanadine salts or peracetic acid solutions) and not noticed a drop off in the PCR quality of DNA retrieved. I guess this might not be long enough to inactivate Brucella though? Its not an organism I have had much experience with
Simon
minsing the tissue firstly and carful add lysozome and protenase k able to kill and destroy the bacteria and extracted DNA: Please look to the High Pure Kit protocol contains all information about the extraction of DNA from different field materials including tissue. If you can not find I can send you one.
Hi Gamal,
Thanks for your response. We did look at lysozyme and Pk. However the number and mass of the tissues made this financial un-doable. We were looking at over $30,000 USD for the cost of Pk alone due to the numerous tissues and their size.
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