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Many colonies but no positive clone - (Apr/24/2006 )

I need help.

I try to clone a 1 kb insert in a 10 kb plasmide.

The insert and the plasmide are digested with BamHI, purified and then digested with EcoRI.
I run a gel to check the digestion.
After purification, I procede to ligation using a 5:1 insert to vector ratio. I have try ligation at room temperature for 1 hour, or cycling 30s at 10°C and 30s at 30°C for one night. I have also try ligation at 16°C for one night.
With ligation at 16°C for one night, I obtained lot of colonies. I use plates with ampicillin and x-gal, since the palsmid bring resistance to ampicillin and have the lacZ gene.
I did colonies PCR on blue colonies and had no positive clones.

What the problem? Can you help me?


I don`t understand why you check the blue colonies. Every colony that grows has the vector (amp resistance). Does the insert disrupt the LacZ gene? Then you should check the white colonies??
You can treat the vector with CIP or Antarctic Phosphatase to avoid religation of vector in case this plays a role as a source of errors here. And maybe use higher ratios of insert:vector because insert is very small in comparison to the vector.