Hi,

first lower the concentration of Your DNA. Normally, You don't want to exceed 200ng/ul of DNA in the reaction for digestion to be efficient. Secondly, I am not sure why You heated the DNA, as enzymes needs double stranded DNA to work on.Also, the long incubation might induce star activity, check with the supplier if this enzyme is fine for long incubations.

Thank you! I am sorry I didn't explain the concentration of my gDNA clearly. The concentration of gDNA I extracted from rat's tail (dissloved in TE) was 850ng/ul. The final concentration in the digestion reaction is 100ug/ul. I have digested the DNA without heat before but it doesn't work well. So I found some suggestions that heating DNA at 65 degree may help so I made a try. But there was no improvement.

it is not clear to me that you have a partial digestion as your 2 cut tracks clearly have the largest dna as smaller ( therefore cut)( and spe is a 6 cutter so some large cut fragments are expected) than the uncut sample but if you are unsure then take an aliquot of the cut sample and add more enzyme and incubate overnight again and run uncudna,first cut dna and twice cut dna . If there is no visible difference between once and twice cut dna then i think that the original digest is fine but if there is a difference then do the first digest again with twice the amount of enzyme. It is often the glycerol that enzymes are stored in that causes star activity and twice the amount of enzyme will not be too much glycerol in a 50ul reaction