filling the digested vector and insert for blunt end cloning - (Jun/02/2014 )
I am going to clone an insert into a mammalian expression vector. both vector and insert contain multiple elements from different sources. So I dont have a lot of options for selecting restriction enzymes. actually I can not find single (or double) restriction enzyme site (either blunt or sticky) which can be used for digestion of both fragments. So I decided to add a RE site, which can not cut my insert, to my primer (like BamH I) amplify and clone it into an TA vector. after sequence confirmation, I will cut the insert with BamH I fill it with T4 DNA polymerase or Kelenow. I will cut my vector with another single enzyme (like Bgl II) fill it and then ligate the two fragment together.
I can not think of other ways but I appreciate any suggestion. please note that my insert is a coding unit for a resistance gene (promoter-CDS-poly A) so I do not need to insert in into a MCS.
Your first part is good, but you should amplify your vector with primers that contain similar BamHI sites, and directly link the two PCR fragments. You can digest the vector PCR product with DpnI to reduce background (use very small amounts of initial vector template in any case). If you put incompatible RE ends on your insert and vector PCR, you can control religation and direction of insertion.
Thank you very much for your suggestion. My vector is too long (12 kb) so I cant amplify it easily and also sequencing of the amplified vector would be really expensive.