DNA Extraction from bacterial cultures - (Nov/10/2010 )
Hi...I am working with bacterial cultures (Staphylococcus aureus and Klebsiella pneumoniae). I first need to extract DNA from bacterial cultures and am currently doing this using a crude boiling method. Downstream applications include screening for antibiotic resistance genes using real time pcr, followed by conventional pcr and then sequencing. My question is: Will the method of extraction I am currently using be okay for all applications mentioned or are there more optimal methods of extraction for each of the applications? Any advice will be greatly appreciated. Thanks.
as long as you are not doing quantification using Real Time PCR, I think boiling approach is still ok.
You can always further purify the crude preps using phenol chloroform extractions, as you would for any other DNA prep.
I am doing the same thing with Escherichia coli. I use a modified rudimentary boiling method which includes dishwashing detergent for lysing and an ethanol wash step. I've been very happy with the results, high yeild DNA, pretty decent quality too. I've run standard PCR, sequencing, and Rep-PCR on these samples and the results are great. As stated above, I would question it's use in real-time but for all other PCR, you should be fine and you could always purify further if necessary.