forgot to digest PCR insert before ligation. - what happened? (Oct/17/2006 )
ok im so stupid. i digested the vector, purified. made insert by PCR, purified and forgot to digest it with the enzymes!!! 
....so i got more colonies on the positive plate and checked them by PCR they are positive!!! 
they contain my insert. miniprep check is however all weird. 
what do you think have happened?
Hi.
Guess you can classify this case as the X-file. I've got plenty of them too but just too little time to seek the answers. 
Cheers
ligation... maybe by strand invasion. T4 ligase is not a clean enzyme.
Hi!
PCR test of colonies is positive not because your DNA was ligated in vector (this is impossible) but because the foreign DNA (contained in the ligation reaction) is spread on LB plate. So when you get a colony you also get a small amount of the foreign DNA which acts as a template in your control PCR reaction! I suppose that the primers you are using are insert specific, right? If you were using a vector specific and an insert specific primer then normally the PCR test would be negative. There is a related paper in the net.
try a analytical digest to confirm the presence of the insert. and since u have a PCR u will sequence it anyway. use primers in the vector to verify the presence of insert.
PCR test of colonies is positive not because your DNA was ligated in vector (this is impossible) but because the foreign DNA (contained in the ligation reaction) is spread on LB plate. So when you get a colony you also get a small amount of the foreign DNA which acts as a template in your control PCR reaction! I suppose that the primers you are using are insert specific, right? If you were using a vector specific and an insert specific primer then normally the PCR test would be negative. There is a related paper in the net.
i also thought of this. however to add to the X-fileness of this case, even PCR of the miniprep turned out to be positive.
Scolix, sequencing can;t be done here. noone is using the sequencer and i am told to only establish the method will take me much more time than i have to graduate...
kathy, what if there r some mutations while u did the PCR. Normally, it should b fine but u never know. And if u had even a single mutation, couldnt affect ur future experiments which u have planned ?
take care and good luck
i know this just seems to stupid that sequencer is there but noone ever uses it.