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How to Test DNA Methylation Status in E. coli Plasmids | Molecular Biology Guide How to Test DNA Methylation Status in E. coli Plasmids Introduction Ever cloned a plasmid in E. coli , only to find your restriction enzymes mysteriously fail? The invisible culprit is often DNA methylation —specifically, modifications by Dam and Dcm methylases . These enzymes add methyl groups to DNA, blocking some restriction enzymes while leaving others unaffected. But how do you test methylation status to confirm it's the problem? In this guide, we'll cover: 3 lab-tested methods to check Dam/Dcm methylation When to use dam-dcm- competent cells How methylation-dependent cleavage (like DpnI digestion) can be a diagnostic tool Troubleshooting tips for failed digests ...

E. coli Dam Methylase and Its Impact on Restriction Enzymes | Molecular Biology Guide Which E. coli enzymes are blocked by Dam/Dcm methylation Introduction: Did you know that 70% of common lab E. coli strains can invisibly modify your DNA, blocking restriction enzymes without warning? That’s because Dam and Dcm methylases add ‘molecular locks’ to recognition sites—and unless you’re using methylation-insensitive enzymes or dam-dcm- cells, your digestions might fail silently. Even more surprising: some enzymes like DpnI require these modifications to work at all. Ever struggled with a restriction enzyme that just won't cut your DNA? The culprit might be methylation —specifically, the work of E. coli's Dam and Dcm methylases . These enzymes modify DNA in ways that can block restriction enzymes, leading to failed experiments and frustrating troubleshooting sessions. ...