I want to insert a gene in a vector using restriction cloning, but the enzyme that I have to use has three restriction sites in the vector. It is imperative that I use only this enzyme and no other, so I can't use a different restriction site or other enzyme. Can someone help with this issue?
I have tried partial restriction digestion with different units of enzyme as well as different time periods of incubation but haven't got a single band. The enzyme is cutting at all three sites whatever conditions I try in the partial restriction digestion.
You can create the mutation on the other two undesired restriction sites with the newly designed primers. Then digest and ligate the gene at the desired site.
You might wish to rethink your strategy. Why must you use this specific enzyme and not some other enzyme for the cloning? Provide us with some additional information and you might get more useful answers because there are likely to be alternate solutions.
As Michael J. Benedik suggested above, in cloning there are always alternative approaches to consider. I myself would caution against partial digestion as a solution, enzymes are very efficient. Even with a short cut time you are more likely to cut at one (or both) of the unwanted sites. Perhaps a map of your vector and insert would be helpful.
I agree that there are always ways around, but if you really insist on using this enzyme, you can do short digest with little enzyme and check it on the gel. If you will see band of the size of your full plasmid (single-cut), that is what you want to cut out of gel.
You will likely see smaller bands originating from double and triple cuts, but as long as there is the full-length plasmid band, you can use it after cutting from gel.
However, even in this band it will be mixture of plasmids cut at the three sites, so expect that only 1/3 of the DNA will be of the right DNA. Depending on where are the other two sites (e.g. in ori or resistance gene), you may expect that only about 1/3 of colonies will be positive.
Anyway, if you need to clone your DNA in correct orientation, it will be only half of the colonies as you are using single restriction enzyme (unless you mean that one of the two enzymes cuts in three positions) and thus only about 1/6 of your colonies will be in your position in right orientation.
Can you start with a different vector? Use TOPO-TA cloning? Use primers that add in a restriction site for a different enzyme for the insert? Site-directed mutagenesis to remove the restriction site in the vector? I do not understand why you must use this exact vector + this exact enzyme.
Something is going to have to be different since your current strategy simply will not work.
Talk with your supervisor, it's a waste of your time to insist you use a protocol that you know will fail.
Enzymes are cheap (mostly), your time is valuable. I'm sure you can come up with a reasonable solution.
- Badges
- Science method