Test of plasmid quality using restriction enzyme digestion, ligation and transfection.

<p>(A) Plasmids purified by glass syringe filters were digested with the indicated restriction enzymes, the digested plasmid was ligated and then re-cut with the same restriction enzyme. 1: Initial restriction enzyme digestion. 2: Ligation at 16°C for 12 hours. 3: Re-digestion with same enzyme. L: 1 kb ladder (Invitrogen) (B) GFP expression following transfection with purified pEGFP-N1. <i>E.coli</i> bearing pEGFP-N were cultured in 200 ml LB and plasmid purified using either a Qiagen Maxi-prep kit or glass syringe filters. The plasmid was quantitated by spectrophotometer and transfected into 293T and HeLa cells using lipofectamine or calcium phosphate. After 2 days, the transfected cells were harvested and GFP expression was analyzed by FLOW.