PHENOL-CHLOROFORM EXTRACTION USING UNPASTEURISED STILTON!

[UGmicro]

I am an undergrad trying to conclude my final year project which was titled 'the isolation and characterisation of non-starter lactic acid bacteria in unpasteurised Stilton cheese'. After carrying out various attempts using a standard phenol-chloroform DNA extraction....it did not work! I was wondering if anybody coulod give me any idea's on why it didnt work? All reagents were new therefore this was not the problem! I was thinking perhaps there is something which usually during pasteurisation is killed but as it's unpasteurised there may be something which is inhibiting this extraction method.
Any suggestions are welcome!

Thanks in advance

[canalon]

I find the idea interesting, but I am not surprised that you did not isolate anything. There are many factors that can enter into account and since I do not know the manufacturing process of the Stilton, I cannot comment on what can happen during production (heating stage that would kill bacteria ?).

However since it is a blue cheese, you should be able to find some Penicillium DNA, so it seems that at least your sampling preparation should be reconsidered:
- Many cheese are acidic, and DNA can be damaged even by weak acid.
- Cheese is fat, and that is not ideal for DNA purification
- What size of sample are you using, maybe it is too small ?
If you can have access to scientific journal I suggest you start by reviewing what kind of protocol have been used for DNA extraction in cheese or dairy, I am pretty sure that you might find some help. A quick search in Pubmed suggest that some steps are added to the regular phenol/chloroform protocol.