I am trying to ligate my insert into the pCDH1-MCS1-EF1-Puro vector from System Biosciences. My insert is 2.7kb and the vector is 7kb. At first I was trying to do a blunt end ligation on the 5' end and a cohesive (sticky end) ligation on the 3' end and tried a insert:vector ratio of 3:1 and 5:1 (Overnight ligation at 14 degrees with T4 DNA ligase) with no success. I then used PCR to add on the complimentary cohesive site to the 5' end of my insert and amplify the fragment and ligate these two pieces together (1h ligation at RT with T4 DNA ligase). At first I was using DH5 alpha competent cells and just recently got some One Shot TOP 10 competent cells. With the DH5 cells I would get lots of colonies/plate (over 100) and the colonies that I tested (~50) did not produce the correct plasmid and ~10 colonies/plate with the TOP 10 cells and again none produced the plasmid.
Can anyone help trouble shoot this with me. I am getting very frustrated and my PI is breathing down my neck because we really need this plasmid to continue our work.
Also has anyone used the pLenti-promoter plasmid from Applied Biological Materials? We are thinking of trying this plasmid next.
Thanks for any suggestions!