HomeFAQsHow many base pairs should my amplicon inserts have flanking the Type IIS restriction site?
FAQ: How many base pairs should my amplicon inserts have flanking the Type IIS restriction site?
Amplicon inserts must posses 5' flanking bases and Type IIS restriction sites at both ends of the amplicon in the proper orientation. We recommend adding 6 flanking bases at the 5' ends of the primers for optimal Type IIS restriction enzyme binding, cleavage, and overall Golden Gate Assembly efficiency. This 6 base pair recommendation supersedes other known end length preferences for the Type IIS restriction enzymes due to Golden Gate Assembly protocols requiring maximum enzyme activity for efficient assembly.
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