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digestions sometimes working,sometimes not! - (Jan/19/2006 )

hi all..

Im havin a problem with my digestion of transformed plasmid with AvrII enzyme (from NEB). supposedly i got 2 bands which are the vector and the insert. But i only got 1 band which was the insert (the correct size of insert).the vector seems 'vanish to nowhere' i dont know...
has anyone know why this happen? and know how to get my lovely vector and insert back?


have you made sure that the size of your insert of vector are not similar and therefore not overlappy on each other on the gel?
If not, are you sure 100% that the site exists on both side of the insert? many times, I get a construct from someone with a certain map, and i keep doing digests and they don't work. eventually it turns out that some sites were lost during cloning but the person who made the construct did not make a note of it. maybe it's a good idea to sequence your construct.
i hope that helps.


Why do you think the standard DNA digest is 20 micro liters? Is there something significant about that number? Or is it something else? Please reply soon!


Trista , I sent you a PM

I do not know if this thread is the best place for your question?