I have two restriction sites which i want to ligate on two sides of my gene of interest. i want to design primers for doing the PCR of the same to ligate. how to make the primers?
you need to add restriction site with extra bases to the 5'end of your primers,here link for this protocol:
https://www.addgene.org/protocols/pcr-cloning/
you need to add restriction site with extra bases to the 5'end of your primers,here link for this protocol:
https://www.addgene.org/protocols/pcr-cloning/
Hello,
You will need to add the restriction site and 5 extra bases as a tail in your PCR primer (the 5 extra bases allows efficient digestion of the linear PCR amplicon). Check this link for different enzyme requirement for additional bp (https://international.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments). I have created a little image that details how to construct this more visually along with some notes on how to create the primers.
Best wishes,
Hanna
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