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problem with cloning p16ink4a into pEGFP-C2 - (Apr/13/2011 )

Hi everyone!
I do cloning p16ink4a into the pEGFP-C2. I digested them with HindIII and SalI in seperate reactions. After that I ligated the vector and insert DNA in ratio of 1ul:3ul; 1ul:1ul; 3ul:1ul (recommendation of Promega); 1V:3V (V vector/V insert) and 1m:3m (m vector/m insert) and set up ligation using T4DNA ligase (16 degrees, 16 hrs). Then I did the transformation into E.coli DH5α. I got a few of colonies on 1ul:3ul; 1ul:1ul; 3ul:1ul plates (A) and many colonies on 2 remaining plates ( B ).
To test colonies, i do PCR-colony with the primers are C-EGFP and SalI. Majority of B have the band at about 540 bps. I pick up the colonies which have bold band and shoot the tip into the tube within 5ml Kanamycin. The recombinant plasmids were extracted by Plasmid mini-prep kit (Solgent Co., ltd) and test by PCR with the primers are C-EGFP and SalI. But i got no band. I test again by PCR colony with the colonies on backup plate and the result is no band I re-do this process many time but the results do not change.
Please give me some advice!!!
Thanks a lot!


The presence of the insert in the transformation mix may be giving your false positives when you pick and PCR off the plate.