I'm hoping to get advice from some more experienced molecular biologists out there...
I am attempting to create an immortal cell line expressing a surface marker, but i have hit a road block. I am using a pLentiTOPO6.3 TA Cloning kit from Invitrogen. My ligations of my PCR insert into the pLentiTOPO vector ALWAYS result in vector/insert constructs with the insert in the wrong direction / orientation. The constructs are grown in Stbl3 E.coli, but I cannot understand why none of my colonies result in a vector/insert construct with the desired orientation of the insert.
My full length insert is 3.7Kb; truncated insert is 3.3kb. I have screened by either PCR, sequencing, or restriction digest about 100 colonies from my several ligation attempts. I have successfully isolated ONLY ONE truncated construct, but NOT ONE full-length construct in the correct orientation.
Your suggestions and/or advice is greatly appreciated :-)