Posted 03 June 2013 - 12:07 PM
Thanks is advance for any help. I am/have been trying to clone a very large transgene (dystroophin) (12kb) into a plasmid for quite a while now and I just cannot get it to work. I originally tried just cloning the whole transgene at once. That did not work, they all went in backwards. After many different strategies and attempts, i was able to clone the first half of the transgene into the vector. The second have has give me problems and now it too as been split into to two pieces. I am at my wits end trying to make this work and would just like to get some fresh ideas. My vector is 9645 bp and the piece I am trying to put in right now is 1797 bp. The plasmid has a Kan resistance. There is also a homologous recombination site in the backbone. I have tried ligation reactions from 1:3 to 1:30, Top10/DH5a/Stbl3 cells, both invitrogen and roche quick/and long ligases. I have also tried electrocompetant cells. Thanks again for any anwsers!