troubleshooting Rsa-I restriction digests - (Jul/12/2005 )
I seem to have a hard time getting a full digest of some fairly large chunks of DNA into ~500bp DNA fragments using Rsa restriction enzyme.
It consistently appears as a partial digest.
I just did a RsaI digestion of PCR product today. In a 10 ul reaction, I use 1 ul RsaI (from Promega), 1 ul BSA, 5 ul PCR product (without any purification). I got complete digestion after 2 hours incubation at 37C.
Are you sure your enzyme is still working? Adding BSA can increase cutting efficiency.
I used NEB enzyme, and no BSA, also, I purified, and had 20uL of DNA together with 1uL enzyme.
I'll try with BSA, but the enzyme appears to be fairly fresh (not older than 6months)