Try to start purimycin selection 72 hours posttransfection with half of all cells that have been transfected. Try to make RNA and protein extracts from the other half. Run RT-PCR and Western blot to confirm transgene exression. The cell with puromycin selection on can be grown in normal medium after 10-15 days drug selection.
So, you try to clone 3-5 kb blunt ended DNA, but people do not perform in this way as blunt-ending ligation could be very poor for 3-5 kb fragments, cloning efficiency could be lower than 10%. You need to try other protocol for cloning.
In your EMSA experiments, I do not think there is any problem in signal developing reaction. The problem is that the shifts could be confirmed in competition assay. The protein and DNA probe associations are in low affinity and non-specific. I am going to forward to you my reaction buffer for reference. After almost 25 years, I can only remeber tha
Usually 1 kb or so can be easily cloned, but quite hard for 2.7 kb in T/A cloning. Covaris片段打碎仪打碎的片断（大小3-5K), what are the ends of these broken DNA fragments, stick or blent-ended? Without knowing what the ends are, how could be the ligation with the vector?Sau3AI酶切产物（DNA基因组）（条带弥散，大多集中在3-5K）, if your vector is not cut with BamH I or Bgl II, t