cloning - cloning in pet28a (Sep/07/2005 )

i was facing a strange problem,whenever i did cloning in pet28a vector,colony pcr will give me the right product but coming to insert release,
plasmid isolated will run above the marker and that will not get digested .what could be the problem ? did anyone come across such problems.

These are two reasons I could think of:

1. Some restriction enzymes are inhibited by dam or dcm methylation, check if the one you are using is one of them.

2. Are you sure that the enzyme you are using is still working, check on another plasmid

I'm not familiar with the pet28a vector - what is it used for and in which bacteria do you grow it?

Do what LeserattePD said, but also check the following:

If single digest: You may have desroyed one of the ligation sites. Sequence if you can.

If a double digest: Check buffer compatibility for double digest w/ enzymes together. Or try a sequential cut in specific buffers.