Restriction digest - (Feb/13/2006 )
I need help!!!!!!!!! 
I'm trying to digest a PCR (5000bp) product with EcoRI but, after digestion, I can't see the lower bands because I have so much smear!!!! How to eliminate this smear??? Thank you!!!!
I'm trying to digest a PCR (5000bp) product with EcoRI but, after digestion, I can't see the lower bands because I have so much smear!!!! How to eliminate this smear??? Thank you!!!!
I would try cloning it into a T-vector first, propagating that and then digesting the plasmid prep.
Thank you!!!! I'll try!!!!! 
if that doesn't help, have you ruled out star activity? what are your digestion conditions?
I use 25 microlitres of my pcr product and I add 0,25 microlitres of EcoRI ( 2.5 U). I put this mix for 18 hours at 37°C. After digestion i make an agarose gel (0,9%) and this gel run at 70 V for 2,30 hours. I use 2,5 U of EcoRI because in 25 microlitres of my pcr product I have 500 ng of DNA and in the instruction of the enzyme are raccomanded 5 U for 1000 ng. Thank you very much for any kind of help!!!
please check this link
please note the section on contributing factors
when you set up the digestion, did you just put some enzyme into your PCR mix? the conditions may not have been optimal for that enzyme and I think perhaps this may be a factor.
Thank you for informations. I read the document on star activity and I'll try some change in my digestion!!When i'll have news I'll tell to you!!! Thank you very much again and sorry for my bad english.