Problem in plasmid digestion after transformation - (Mar/10/2014 )

Dear Friends,

I encountered a problem related to digestion of transformed Pet22b, while after transformation screening of the colonies revealed positive transformants but to confirm the transformation purified transformed Pet22b was subjected to digestion by NdeI and XhoI for 2 hrs, i am using THERMO enzymes i checked the compatibility of the enzyme buffers and used Buffer O, on running the digested product on 1 % agarose gel there appears no digestion whatsoever, i also did PCR of the purified transformed plasmid and my gene was amplified i cant resolve the problem with the digestion and release of the gene, do guide me with this

i am using NdeI and XhoI

A common problem is the presence of inhibitors in your DNA. Ethanol, and (especially) Gu-HCl from miniprep buffers and wash can prevent digestion. This is often made worse by people deciding to do restriction digestions in low volumes, where the majority of the volume is from miniprep'd DNA. Redo the digestions in >50 ul volume with less than 10 ul of that voume from the DNA. The dry spin of the miniprep columns is critical in reducing ethanol contamination.

I had such a problem before, and after sequencing the insert, I found why!

mutation in one of bases in cutting site.

i did the digestion of 5 ul (300ng/ul) in a total volume of 100 ul but it is still not showing any results probably mutation may be a cause but it would take me at least 15 days to get the sequence, sequencer not available in my lab