plasmids turnedinto smears after NcoI digestion - restriction enzyme problem (Apr/04/2005 )
Hi all.I am frustrated this days for I encountered a strange problem on plasmid digestion reaction.now let me make a description: my plasmid contains only single cutting site of NcoI,but when I conduct the digestion reaction with the enzyme (the enzyme added was already lower than as usual),always relatively low-molecular weight smears yieled instead of the expected single ,linear band! differernt lots of enzyme did not change the result while decreased amout of enzyme or limited reaction time slightly improving the state.
here can any experienced friend give me some advices?thanks in advance!
What you describe is really strange. It could it be that your RE is munching away your DNA. This has happened to me sometimes when using old, long-expired, enzymes: instead of just not cutting the vector, they seem to degrade it. But I always solved the problem by changing the enzyme lot, and you mention that this did not had any effect for you. Have you checked if your vector is behaving in the same way with other enzymes, or do you get the right size band when digesting it with another single-cutter? Is NcoI acting that same way if you try to digest another unrelated vector? Is your vector in water/ TE, or in any other buffer that may affect the enzyme activity?
Sorry I could not be of more help!