At the first step the MRSA colonies will be selected on CHROMagar plates. After that I would like to perform an real-time PCR to detect mecA and femA (only for qualitative purposes).My first choice was actually a simple duplex colony PCR (so I don't have to isolate the DNA), but then I have to run a gel. And it would be simpler and more rapidly to assess the colonies due to their melt curves. Because of the high number of the isolates I am looking for a cheap and rapid method to isolate the DNA. Would the standard rapid lysis method (lysostaphin, proteinase K and Tris buffer) deliver a DNA that I can use in the real-time PCR? Perhaps somebody has already had profound experience in such problems and can help me? I would be grateful for any advice. Many thanks in advance.
The method I mentioned above:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC265364/pdf/jcm00031-0083.pdf
I used melt curve genotyping on crude plant DNA extracts. I just had to dilute my DNA samples to reduce the concentration of "gunk" (cell wall debris, membranes, etc). I think I used a 1:50 dilution. Try it and see!
I am pretty sure that it will be work with lysostaphin and proteinase K. But lysostaphin is relative expensive (1 mg costs 107 EUR by Sigma) and this amount would be enough for only 200 runs. And unfortunately by MRSA it cannot be replaced by cost-effective lysozyme, because of their resistence. I found another protocol for S.aureus Colony PCR (http://richardsonlab.web.unc.edu/protocols/colony-pcr-for-s-aureus/), where they use following lysis buffer:
20 mM Tris (weighed out, not pH’ed)
3 mM MgCl2
0.5% Tween 20 (v/v)
0.5% NP-40 (v/v) (we use Igepal CA-630)
60 µg/ml proteinase K (final con’c)
Perhaps somebody has already used this protocol for real-time PCR?
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