Am working with cloning, and plasmid has an Sap1 restriction site at Forward and reverse (purchased the cut plasmid from an company, they call it Electracloning) and the insert gene has forward and reverse ends of Sap1. After cloning the gene into the plasmid, Sap1 restriction enzyme will not cut the plasmid in Tango1x buffer, my question is how to make Sap1 cut the circular plasmid, what changes must be made for the restriction enzyme Sap1 to work ?
Thanks in advance.
Because it is a continuous circular and it does not have specific nucleotidebase intercept to act on!
Without knowing the sequence of the vector you are using it is hard to predict. But SapI is an asymmetric enzyme that cuts away from the recognition site. So if the recognition sites were located in the stuffer fragment then the recognition site might have been lost upon creation of the clone.
Alternatively there may be an issue with your DNA or your enzyme. Try a different enzyme on your plasmid and the enzyme on a different template and see if those work or not.
Linearized plasmids which I came across during my work, would loose the SapI site after getting ligated with gene due to their design. I would suggest you to confirm the vector sequence design before concluding anything regarding non functioning of SapI enzyme
- Badges
- Science topic