How do I calculate how much DNA I need to add to a ligation reaction?
So, for typical cloning applications we recommend 50 ng of the vector, which will be 25-30 fmoles depending on your vector size. For inserts of similar size to the vector, we recommended a 1:1 molar ratio of insert to vector. If your insert is smaller than the vector, say if you’re trying to ligate a 1kb insert into a 3kb vector, you’ll need a higher ratio, in this case about a 3:1 molar ratio of insert to vector. If your inserts are very small, even higher ratios may be needed, sometimes as high as 20:1.
You can calculate the amount of DNA you need for your specific reaction using the NEBiocalculator on NEB.com.
And remember, if you have any addition questions, you can always contact us via email at firstname.lastname@example.org.
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