Hi, I wonder what factors could lead to failure in the r.e. digestion
of lobster genomic DNA. I was trying to digest 5ug DNA with 50 U HindIII
and 20 U XbaI but without success, thought the digestion on the vector
was all right. I did the following trials after the first failure:
1. Reduce the glycerol level from 14% in the first trial to 5%.
2. 65C bath of the DNA sample before addition of enzyme, to destory any
dirty stuffs that might affect the enzyme activity (the bath was 30min)
3. millipored the DNA sample so that any salt present could be removed.
The DNA smaple was prepared by proteinaseK/phenol/chloroform/EtOH ppt.
Any suggestion would be much appreciated. Thanks.
Dept. Zoology, U.Maine