Can I use EcoRI and NheI in Saccharomyces cerevisiae

[Hiro Protagonist]

Hi all

It seems like kind of a stupid question to myself, but I am somewhat stuck at the moment. I aim to cut a specific protein out of the whole genomic DNA of S.cerevisiae, with primers wich contain overhanging recognition sites for EcoRI and NheI, and clone it into an his-tag expression vector. What currently confuses me is that these restriction enzymes appear to be able to mess up the whole genomic DNA, or are their recognition sites also methylated in S.cerevisiae ?
So if I don't want to get the whole DNA cut in a thousand parts, am I supposed to only use enzymes, which occur in yeast naturally, or are there better solutions than that?

Thanks in advance

[Bryan Jones]

Why are you worried about messing up the yeast's genome? The yeast is already dead by the time you are copying genes out, so it won't mind having it's genome messed up.
If you are concerned about the carryover of genomic DNA for the next cloning steps, then I think what you are missing is that you use the primers to amplify your gene of interest out of the yeast genome with a PCR reaction. At the end of your PCR reaction, you will have millions of copies of your gene of interest for every piece of genomic DNA. At those levels, the genomic DNA isn't generally a concern for basic cloning since there is so much more of your product. 
If you are still concerned about genomic/template DNA carryover, using something like a QIAGEN "QIAquick PCR purification kit" (or any of the cheaper competitors) should remove most of the large genomic DNA.

Hope that helps

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[Hiro Protagonist]

Yeah thanks, I kind of messed up the fact that the polymerase will only anneal the DNA at the sites which the primer is bound to and not everywhere where it is cut. So if I clone it in a first step I probably shouldn't have any problems with the rest of the genomic DNA, guess I didn't think of that because it was in the middle of the night and I was really tired😅, but thanks again ^^