pRL-CMV vector - 2500nt insert. Is it possible? (Dec/10/2010 )
I would like to clone my insert into pRL-CMV vector from Promega
but I wonder is it possible to clone a quite big insert (2500nt). I can cut CMV Promoter,intron and luciferase gen with BglII and NotI and clone my insert so the lenght of vector with my insert would be more or less the same like original. but is it safe to replace almost half of vector with insert?
I just want to clone my insert and after that only obtain RNA from in vitro transcription. I dont want to do anything else with it.
any halp will be appreciated
2.5kb is small. Don't worry.
Big is cloning 190kb DNA fragment.
The only essential part of the vector is the selection marker (which you can replace) and origin of replication (ORI). Leave those two alone and the plasmid will replicate okay in bacteria.
I am assuming that your insert has its own promoter? You still need a promoter for in vitro transcription. T7, T3 or SP3 promoter depending on the viral polymerase that you are using.
I will introduce T7 promoter into my insert before cloning. I appreciate your help:)