I have followed suggestions from experienced researchers to try and get gram-positive DNA by extracting with the QIAGEN DNA Mini kit, but even with a 10 minute centrifugation at 13.000 RPM before starting the manufacturers protocol (discarding the supernatant and using only the pellet), I could not get PCR amplification from the clinical samples extracted. Any suggestions?
I would probably use the old fashioned way i.e. lysis with SDS and lysozyme followed by phenol chloroform extraction. With the gram positives I would increase the EDTA concentration ten fold over the classical methods. They tend to have a lot of nucleases and when you sheer up the genomic DNA is gets chewed quickly. I'm not sure if the qiagen kit is amenable to adding more EDTA (which chaelates the Mg+2 ions and therefore slows nuclease activity a lot).
Many thanks for your suggestion, Boris. As I'm running real time PCR, is lack of purity not going to be an issue with this extraction? In case not, would you mind suggesting me a detailed protocol for this extraction? Best wishes, Daniel.
We have received some samples from DNA Genotek and related products from different companies for testing, but the prices in Brazil of this type of media is prohibitive (especially because we would need almost 300 of those). We therefore collected whole saliva samples in falcon tubes.
New England Biolabs has an enrichment method for bacterial DNA that relies on selective methylation of bacterial versus genomic DNA. The procedure was published in a recent Genetic and Engineering News by NEB scientists. A full publication describing the procedure is also available
Many thanks for your suggestion, Paul. I'm going to consider that option. Best regards, Daniel.
If you have any difficulty finding the paper, please send an e-mail to me at [email protected]. I think I have an electronic copy I can send to you. Best Wishes
Paul
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