Digesting ethanol precipitated DNA with AseI - (Jun/28/2008 )
Hi, I am doing PCR and then ethanol precipitating the PCR product and then digesting it with AseI. It doesn't seem to cut. Is AseI very sensitive to trace salts and/or ethanol that might remain after the precipitation?
I don't think there is anything special about AseI.
1. I guess you are doing 70% wash.
2. If no, do. If yes, do twice.
3. Check the sequence of your DNA (electronically and by sequencing) to make sure it has AseI.
4. Use a different tube of AseI (expiry, poor storage)
5. Check buffer, temperature etc.
6. Are you directly etohing your PCR reaction? Why not PCR kit purification or Phenol/chloroform/etoh?
7. Check with some other enzyme, if it does work, may be buy a new AseI.
Check the NEB website for the details about Ase I. If there's anything unusual, it'll be there. Or ask the manufacturer for specifics.