Problems with restriction enzyme digest. - (Jul/29/2007 )

I have cloned my desired insert ( which contains specific restriction enzyme sites ) to my vector. After transformation I obtaied colonies on the plates. The colony pCR looks good, but once I do restriction enzyme digest with Nde1 and Bam H1 I have not been getting the desired bandings. And none of the other restriction enzymes work as well.,on any of the colonies that I have tried so far. Almost 12!. Is there anyway that I could check that the colonies that I got on the plate contains my desired insert, or is there any other way to confirm that I have the proper clone. :blink:

Thank you,

A restriction digest on miniprep DNA is a very definitive way of telling if you do have the clone. That is about as sure as you can get without doing a sequencing.

I am however curious about about the colony PCR. Did the reaction amplify across the junction between the vector and insert @ one primer binds to the vector and the other vector binds to the insert. Colony PCR is sensitive enough to detect leftover insert DNA from the ligationi reaction that is sitting on the plate. Thus using primers that amplify only the insert (not the junction between insert and vector) will always show a signal.

Concerning signals... a real positive signal for colony PCR is a huge big black band. It is very intense singular band, rarely if ever are there secondary bands, as the real PCR product will have consumed all available dNTPs during its amplification. The signal is very very apparent. It is a kind of signal that leaves no room for doubt or second guess.

If you are unsure if the band is real, it almost certainly isn't real. Such is the strenght of a real signal from colony PCR.

run uncut miniprep DNA along side the original vector. A size shift in gel might indicate an insert but not conclusive.