Hi Fleur,

I am assuming you are doing reduced representation type experiment as these two enzymes are typically used for assaying methylation across the genome. HpaII is sensitive so it will not cut methylated CpGs in its restriction sequence. MspI will cut its sequence regardless of methylation. My only suspicion here is that there would be some type of contaminant, but sounds like you already tried dilutions? Try reducing the DNA to 25% of the reaction volume or less and scale the units of enzyme based on the amount of DNA you are trying to digest. Maybe you could divide up the human sample into multiple digests with extra water and BSA to see if this helps. If both enzymes are not cutting the DNA and you still have a high molecular gDNA band on a gel my bet is on some contaminant that is still there, maybe high salt.

In my experience digesting human DNA with HpaII will show nothing on gel (in terms of short fragments), however they are still there.

If you suspect inhibitor, I will suggest taking 10% of the reaction in new tube and adding some 100-200 ng plasmid that will or will not produce digestion fragments. If the plasmid is not digested, then you have inhibitor and enzyme is not working. I will second Kenneth - add extra BSA . Hope that helps.

I agree with the previous posts, contamination of some kind of inhibitor is the most likely culprit. Petre's plasmid suggestion is a great way to test for this. If you do have contamination I suggest doing a simple AMPure clean-up to purify you template DNA. This is what I did when I had the same problem with a double digest and it worked great, however any kind of DNA clean-up should work. Good luck.

Thanks all for the input. I also suspect there is some kind of contamination. I'll test your suggestions.