unique restriction sites - (Aug/12/2005 )
Hey guys,
I want to do some cloning. Unfortunately the 14 kb plasmid hasn't got any unique restriction sites at the area of interest. I had this idea to modify this plasmid and produce a subcloning plasmid for my purpose. The idea is to cut the plasmid with a restriction enzyme that cuts at position 7000 and 13500. I would purify the 7.5 kb fragment containing the origin of replication and would ligate it. This would give me a smaller plasmid containing the desired unique restriction sites in the area of interest. I would use this vector just as a subcloning vector not as a expression vector (also I would loose the stopcodon with this procedure).
Just try to get some feedback from you guys.
Is there anything I have to be aware of?
I want to do some cloning. Unfortunately the 14 kb plasmid hasn't got any unique restriction sites at the area of interest. I had this idea to modify this plasmid and produce a subcloning plasmid for my purpose. The idea is to cut the plasmid with a restriction enzyme that cuts at position 7000 and 13500. I would purify the 7.5 kb fragment containing the origin of replication and would ligate it. This would give me a smaller plasmid containing the desired unique restriction sites in the area of interest. I would use this vector just as a subcloning vector not as a expression vector (also I would loose the stopcodon with this procedure).
Just try to get some feedback from you guys.
Is there anything I have to be aware of?
Dear freind,
I got an alternative way of doing. You may linearlized the plasmid at the disired location with RE. This will produces a sticky end at both site of your plasmid. Later perform a long PCR using high fidelity Taq (Roche) and primer with you disired RE site incorperated. Since you are using a high fidelity Taq, you will get blunt end PCR product. Later Ligate you PCR product with T4 ligase. By doing this, you can replace any RE site with another RE site you want at the sametime you maintain your plasmid as a expression vector.
Best regards
Hadrian
Don't ignore the possibility of PCRing out your vector backbone. This is likely easier than messing with obscure restriction enzymes, and you'll get the piece you care about. Fidelity of the vector backbone is not usually important, and you have a strong selection for functional plasmids. Just a note: Taq leaves overhangs, even when Roche makes it. I think what you meant was that other enzymes (Pfu, Pwo, Phusion) leave blunt ends. Even the mixtures of Taq + other enzyme leave overhangs (e.g. Invitrogen Platinum PCR supermix high fidelity).