I want to do plasmid extraction from Staphylococcus aureus for plasmid fingerprinting.
I would recommend you to do a midi preparation to have enough DNA available for your purposes. What kind of plasmid do you want to extract? Low-Copy? High-Copy? For high copy plasmids, a 50 ml culture should be enough. In the other case, use 100 ml cultures. For this purpose, you will find enough kits available on the market. We are using kits from Quiagen, but I don't know if these kits are available in Iran.
To extract the plasmids from Staph shouldn't be a problem. I will recommend alkaline lysis method athough you will need to include lysozyme but if you have kit it is also good. My problem with kit from some companies is that sometimes those buffers might not be good enough.
Thanks MR Dogan & MR Bamidele;I tried extraction before by alkaline lysis method and lysostaphin but I couldn't get any result.....there is no special kit for gram positive bacteria, all are for gram negatives! and I want to extract whole plasmid in order to plasmid fingerprinting without genome contamination I didn't have any knowledge about their copy number!
The Quiagen kit we are using is also based on the alkaline lysis method. How much (Volume? Concentration?) of the lysostaphin did you use? From which company do you get it? In the case of S. aureus, you have to use lysostaphin for sure.
Lysozyme doesn't work, because of its O-acetylation of the peptidoglycan (C6 atom of the MurNAc), which confers resistance against lysozyme.
The best thing would be to write down your protocol here, so that I could see, what you are doing differently than me to give you optimal advices.
you can use qi*gen or another kit for plasmid extraction, the important think is how to break cell wall of S. aureus. i have experiences to combine enzymatic lysis buffer (you can look the compotition of enzymatic lysis buffer in DNe*sy blood and tissue kit handbook) and lysozyme 20mg/ml, it's work. or you can combine enzymatic lysis buffer with lysostaphin
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