I spent 1month trying to clone a insert into a vector that we wanted to use for an over-expression study. Basically, it just didn't work. So we tried TOPO and voila.... it worked fine within 1week!
I have now digested the TOPO-vector (carrying our insert) with EcoR1 and got bands as were expected (our insert carries 3 EcoR1-sites within it).
But the PCR-produced insert that we cloned into the TOPO-vector is also flanked by EcoR1 on both ends of it, and will after digestion with EcoR1 create 2pieces of 13-18bp fragments (plus the 3 bands created by EcoR1-digestion within the insert itself).
But these 2 fragments don't show on the electrophoresis. Is that because the bands simply are way too small to be visualised?
Now I have been told to digest the TOPO-vector (that carries our insert) with restriction-enzymes (for the primers that we used for amplifying the PCR-insert).
This digestion will basically cleave out our insert from the TOPO-vector. Then we will send it all to sequencing.
Isn't it a bit counter-productive to first PCR-amplify a insert, incorporate it into a TOPO-vector and then cleave it OUT AGAIN???!
What is this purpose for?
Can you use a TOPO-vector (pCR4) for an over-expression study? If yes, does that mean that I now need to design primers for the promoters that it carries?