I am using two REs enzyme (EcoRI and NdeI) to cut my plasmid. At first, I used two enzyme together and one of them did not worked. By an initial search, I found that it is a common problem and if I use them separately, the problem will be solved. But, still I have problem with EcoRI. Then, I got suspected to my EcoRI enzyme about being unfunctional. so, I borrowed enzyme from three other friends but still something is wrong with the experiment. In the last step, I checked my buffer by using a fresh buffer from a brand new kit and you can guess how it worked...

Finally, by checking the EcoRI specification, I found that it is sensitive to "CpG Methylation: Blocked by Some Combinations of Overlapping" and I am thinking this is the problem regarding my previous experiments.

So, anyone know how do I can stop methylation on my plasmid in E. Coli (except changing the E.coli strain)?