I am designing a cloning experiment to remove a domain of the protein my lab studies using gibson assembly. It seems to me that the overhangs I design into primers targeting the two sides of the domain (essentially bridging the gap between the two ends, beginning just before the domain and continuing just after it ends) will likely anneal to both sides nonspecifically, resulting in a mixture of products, some that contain the domain I am trying to remove, some that don't.
Are there any measures I can take to prevent nonspecific annealing such as digesting the protein beforehand or manipulating the Tm values of each 'segment' of the primers? Is there a better technique that I can use to eliminate the domain?
Thanks!
You might want to add more information on how you are doing this. Are you digesting the vector in a way to remove the domain then re-bridging? also you if your plasmid is small enough you just amplify from both sides of the domain then do blunt ended ligation after phosphorylation without the need for an assembly.
ah, good point! I had been intending to digest and re-bridge. The plasmid 3.5 kb and contains a 3.5 kb insert, from which I want to remove roughly 100 nt. I am not sure if this is small enough, and would love some input!
This is fairly a small plasmid a ~7kb amplification should not be difficult with polymerase like phusion. All you need to do is make two primer that point away from each other and leaves the 100bp you want to remove out of the amplification. Do DPNI digestion and gel purification to get rid of the parent plasmid and then use t4 kinase to phosophorylate and ligate.
P.S you can simplify this by ordering phosphorylated primers then you can skip the T4 kinase step.
Thanks for the insight! My question is, how do I control the polymerase? It seems to me that 100 nucleotides is ultimately not very large, and the polymerase would simply proceed to the domain I am trying to remove and amplify it from both ends.
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