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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bqifmubn

Authors: Addgene The Nonprofit Plasmid Repository

Summary: This protocol describes ddPCR titration of AAV vectors. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/aav-ddpcr-titration/.Sample Data: When analyzing data there should be a clear distinction between negative droplets (black) and positive droplets (blue).The no template control (NTC) should be close to zero (B08). At Addgene, runs with an NTC >5 are invalid.To reduce NTC values, we recommend wiping down all pipettes and equipment with 10% bleach prior to use and keeping all reagents and samples on ice or pre-chilled 96-well freezer blocks during use.In this protocol, a dilution series is prepared for each AAV sample and the 3 final dilutions are assayed. The samples that are assayed are diluted 2-fold serially therefore, the concentration obtained by ddPCR should decrease by a factor of 2 across the dilutions. In the example below, 2-fold serial dilutions of a sample were loaded in wells A04, A05 and A06. As shown in the image and table below, the concentration of positive droplets decreases by a factor of ~2.To increase the accuracy of the titer, calculate an average of several dilutions. For additional tips on AAV titering using ddPCR, read our blog post.

Affiliations: Addgene

External URL: https://www.addgene.org/protocols/aav-ddpcr-titration/

Version: 2

Publication Date: 2020

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