Okay last time it was a E-PCR question this time its about PCR hehe.
I"ve got a gene that I want to cut out of its present vector and ligate it into a GFP vector. Now i’ve designed primers already to amplify the gene with the restriction sites but the stop codon is the problem.
The PCR is all well and good but how can I ligate it into my GFP vector and make a fusion protein with GFP and my protein if there is a STOP codon.
You can simply design out the stop codon, but I don’t know how you’re trying to do the construction. Whose stop codon is in the way, GFP’s or your protein’s? In other words, is GFP the 5′ end of the proposed fusion, or are you trying to put GFP downstream of your protein?
Exactly how you do it will depend on what’s already in the vector and which way you’re trying to make the fusion. I’m guessing you already have a GFP-expression vector and the question is, how to put the protein of interest as a C-terminal fusion in-frame with GFP?
I’ve actually got it figured out. Its the stop codon of the protein and GFP is fused to the proteins C-terminal end.
When I was designing the primer I just left that part (stop codon) out of the primer and started it one codon before the stop.
Then added my restriction enzyme site after that.
Then made sure it was in frame with GFP. But because my protein needs to be in frame from its C-terminal end with the GFP only the reverse primer was determined to be in frame. And any bases that were added in order to make the protein into frame with GFP was added before the restirction site ie. right after the last codon of the target protein (otherwise it will be cut off ;)).
Thanks for the insight blcr11.
Hope this helps some others out?!?