How can I delete a fragment that is not surrounded by restriction sites? - (May/03/2007 )
I want to remove a 300bp region from my plasmid vector, but this region does not have restriction sites anywhere near. What is the easiest solution to delete the 300bp region? Can I insert extra restrction sites or should I amplify the plasmid-minus-300bp-region by PCR?
My vote goes to PCR.
As alredy mentioned, amplfying the entire plasmid via inverst PCR is possible but how easy said task would depend on the size of the plasmid in question. If it is below 10kb, it is doable. Below 7kb it is both doable and easy.
Another alternative is to combine both PCR and restriction digest. This idea is far more complicated and messy then the simple inverse PCR idea.
In this idea, you PCR up the region of interest (minus the 300bp) up to the closet unique restriction sites.
Then using the same restriction sites, cut out the region between the unique restriction sites and replace it with the PCR product (which has the 300bp truncation).
To make the trucated PCR product you will have to either fuse 2 PCR products, the segment infront of the deletion and the segment behind the deletion. Or use bridging primer mix... it contains primers that have homology to the sequence infront and behind of the deletion. PCR using this mix results in the sequence in between the homology to be deleted.
Or you can design primers with a RE site in them, use them to amp the plasmid then digest and ligate back together
design primers to construct a restriction site is the quickest way i think
sometimes we need to construct site in promoer regions cause its not a good idea to delete about 300 bp from a promoter