verifying a positive transformant - (Sep/04/2010 )

Hi everyone,

I wanted to sequence a size selected fragmented cDNA library (150-350 bps). I ligated the fragment into a PCR blunt vector (Invitrogen) and transformed into a DH5alpha1 cells. I did a blue-white screening on LB+Amp plates. Now my question is how can I identify the vector with my insert of interest? Do I randomly select a restriction enzyme and see if the restriction pattern is different between blue and white colonies. The fragmented cDNA library has been created from total RNA. Any kind of help would be really appreciated.

Thank you!

Colony PCR? Have one primer in the plasmid, the other in your target gene and amplify - ones which don't amplify don't contain the target gene.

bob1 on Tue Sep 7 00:59:33 2010 said:

The classical approach would be a colony blot followed by hybridization with a labeled probe. These days, if you just want sequence, it might be more effective to sequence your entire library and select the piece you want. You could then synthesize it rather than isolating it.