Restriction digest - (May/03/2003 )

Dear all,
At the moment I have a problem with my restriction digest as the enzyme failed to cleave. I am using PflMI (from NEB) to digest PCR products and used lambda DNA as control. I followed instructions supplied regarding incubation and buffer used. The reaction was supplimented with BSA and left for 18hrs. This enzyme is sensitive to Dam and Dcm methylation. Any suggestions will be appreciated. I was going to decrease the amount of DNA used for digestion, using only 10ul PCR product in a 50ul reaction volume.

Thanks

dnalabmalta@yahoo.co.uk

how about ur control? If u r digesting an enzyme site designed in PCR primer,
it's probably because that ur protection bases outside of the enzyme site is too
short.