I'm planning to perform subcloning and need to include restriction enzyme sites in the primers to amplify the insert. In addition to the restriction enzyme overhang on the forward primer, I also need to add a GS linker on the 5' side of the forward primer. How many nucleotides can be added to the 5' side of the forward primer, aside from the restriction enzyme overhang? Any suggestions would be greatly appreciated. Thank you!
1/ As many as possible (with respect to what is possible to synthesize). This may be used to introduce e.g. affinity tags or sequences overlapping with other genes to create gene chimeras by PCR.
2/ There is also a minimum number of nucleotides you should add at the 5' of the primer before the restriction site. These nts are needed for the restriction enzyme to function efficiently (e.g. EcoRI typically requires at least two bases on each side of its target sequence to cleave efficiently). This number is restriction enzyme-specific, and is specified by (some) manufacturers, such as NEB.
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