sticky question for people who work with Staph? - pretty please with cherries on top, help me? (Jul/31/2006 )
So I have a question for those of you who have worked with S aureus, and I hope you can help me? 
over the past three years I've done a variety of projects using 3 or 4 different strains. I haven't needed gDNA often, or very much. each time I have done a 'rough' prep using lysostaphin and proteinaseK, followed by phenol/chloroform prep to clean up...I've posted it on here a few times for other people too.
although this gives me plenty of DNA, I don't try to store it long because it's not really a very good prep, I would just set up a PCR or two and toss it out. the prep itself is cumbersome, but again I haven't needed it much so it wasn't a big deal.
well, now I will be screening many clinical isolates for a particular pair of genes...like, many many...several hundred easily, possibly a couple thousand in total over the next few months. the thought of doing phenol/chloroform preps on that many preps makes me cringe. so, I ordered a bunch of wizard preps, thinking I would use the gram positive version...I've been working on it a few days now, and have been in close contact with Promega's tech support, and haven't been very successful. are there unwritten rules to this protocol??? for those who may be intimately familiar with the gram-positive variation on this protocol, let me tell you what I've tried, and what I think might be happening...but if you have a way to make this protocol work properly for S aureus, please let me know!!
so, first of all, I've remade the EDTA and doublechecked the lysostaphin concentration, so it's not the reagents that I supply that are the problem.
second....if I add the lysostaphin in the recommended amounts, with or without the lysozyme, it's WAY too much and the cells lyse completely in about 11 to 12 minutes.
even if I stop the lysostaphin before the solution clears (and I have attempted timepoints, using vastly less lysostaphin than recommended and no lysozyme), when I go to pellet the mix, even if I spin it for 5X the recommended time, I don't get a discrete pellet...there's a viscous glob of clear stuff and there's no removing of any supernatant, I don't care what they say...so I've been stuck here at this step, worried that the EDTA in the initial resuspension buffer will mess up downstream parts of the protocol, but unable to get a pellet that can be resuspended in the nuclei lysis solution
ANYBODY WORK WITH THIS KIT FOR THIS PURPOSE AND CAN HELP ME? PLEASE PLEASE PLEASE?
The last thing I can think of to try, is just to run it through with the EDTA, skip the nuclei lysis buffer step entirely, and hope I have clean DNA at the end...I am trying that this afternoon, but if anyone has a method that's proven, please let me know!!
thanks
A 
I assume you are using the Wizard genomic DNA kit, and not the miniprep -- is that right? I have not used that, but have used the QTIP Qiagen kit, which worked well for my organism, but it was not a Gram+.
yeah, the genomic kit
it was designed to isolate gDNA from WBC's, but there are adaptations for gram-, gram+, yeast, mammalian, etc
I was trying to come up with the easiest way to get the gDNA without having to do P/C extractions on tons of samples *sigh* now I've got two kits, good for 1000 preps...need to figure out how to make it work
I have pulled up a handful of papers, but have found little specific detail...one of those things everyone knows, so no one mentions specifically what they do 
All right, I have found a few contacts from relatively new papers who use this kit, and am emailing the authors....hopefully I will hear soon!
How will you be screening them? If it's by PCR, I wouldn't even bother trying to recover genomic DNA first -- just do a colony PCR.
Homebrew, we thought of that, but it is problematic...occasionally a colony PCR is not successful with Staph because of the rough nature of the prep (I believe it has to do with cracking the peptidoglycan and separating it from the internal stuff) I need to know definitively if the gene is there, for statistical purposes. So, we can't be repeating negatives that may or may not really be negative or we'd spend a lot of time chasing our tails. We want to start with good template to begin with so we don't have to go back.
What about if you resuspend the cells in dH2O, perhaps with some lysozyme, then place them in a boiling water bath for a while, briefly spin the junk out, and take an aliquot of the supernatant? Hard to believe there wouldn't be DNA there adequate for PCR.
But, then again, I don't work with Staph... 
you can't get it to work with lysozyme, not like e coli (I sometimes screen transformants with your method 
)
you have to use an enzyme called lysostaphin (sometimes both if it's a strain with particularly thick pgn)
and about 20% of the time, you don't get good clear PCR results, probably coming back to proper lysis without degrading anything...it's a real pain. I like staph, but I used to work on gram - bugs that were much easier to deal with 