Hi all,
I have been cloning certain transcription factor subunits in Pgem-Teasy vector. However I am facing a huge issue with one factor when I am amplifying it from cDNA. After doing a touchdown PCR, I have gel eluted the 512bp fragment using MN gel purification kit and checked for its concentration and purity using nanodrop (40ng/ul and 1.8). After using 1:3 and 1:5 vector:insert concentration, I make a 10ul reaction and keep for overnight incubation at 4'C and 16'C. The next day, after transformation (basic protocol) I follow up with a blue white screening. I get tons of white colonies on both my plates. Even though PCR shows all positive for my GOI, digestion shows null results. I am using anew kit of PgemT . Suggestions?