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.
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#2
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Legendary Graduate Beggar
Posted 10 November 2010 - 08:48 AM
as long as you are not doing quantification using Real Time PCR, I think boiling approach is still ok.
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..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn’t kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn’t kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
#3
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Hmmm, I think it's working
Posted 10 November 2010 - 03:54 PM
You can always further purify the crude preps using phenol chloroform extractions, as you would for any other DNA prep.
#4
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Posted 16 November 2010 - 06:08 AM
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.
