no restriction EcoRI in genomic DNA - (Aug/30/2005 )
We are working with an amazonian plant, we wanto do AFLPs, we isolate DNA and we digested the genomic DNA with HindIII and MseI, but when digest with EcoRI we dont have any result, the DNA is complete, there isn't restriction evidence. The EcoRI enzyme hasn´t expired because the positive control (lambda DNA) digest totally, we obtain the bands expected.
So, we want to use the Invitrogen AFLP kit, it works with EcoRI and MseI and if the EcoRi doesn´t work, we can't use it.
We don't know if there are any inhibitors for the EcoRI enzyme in our genomic DNA? if it so, What can we do?
Please really need help any suggestion is welcome!!
How have you determined the DNA is "complete". Did you run it on a gel and see a smear? You would expect a lower MW smear compared to uncut genomic DNA. It should work fine unless your DNA prep is dirty or you forgot to add the correct enzyme buffer, ect.
EcoRI is the only one of the three enzymes you mentioned which would be sensitive to CpG methylation. You might want to look at REBASE for more information about methylation sensitivity of restriction enzymes. http://rebase.neb.com