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Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol goes through AAV purification by Iodixanol gradient ultracentrifugation. To see the full abstract and additional resources, visit the Addgene protocol page.Sample Data
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. AAV Purification by Iodixanol Gradient Ultracentrifugation. protocols.io https://dx.doi.org/10.17504/protocols.io.bawqifdw Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for general transfection. To see the full abstract and additional resources, visit the Addgene protocol page.Sample Data
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. General Transfection. protocols.io https://dx.doi.org/10.17504/protocols.io.bawuifew Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for general transfection. To see the full abstract and additional resources, visit the Addgene protocol page.Sample Data
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. General Transfection. protocols.io https://dx.doi.org/10.17504/protocols.io.4xqgxmw Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for inoculating a liquid bacterial culture. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/inoculate-bacterial-culture/.
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. Inoculating a Liquid Bacterial Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.5abg2an Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for creating bacterial glycerol stocks for long-term storage of plasmids. To see the full abstract and other resources, visit the Addgene protocol page.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids. protocols.io https://dx.doi.org/10.17504/protocols.io.4g5gty6 Copy
Authors: Addgene The Nonprofit Plasmid Repository
Group: Coronavirus Method Development Community
Summary: This protocol describes RNA extraction without a kit. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/kit-free-rna-extraction/
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. RNA Extraction Without a Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.beabjaan Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: Please see the Addgene website for additional details.
Proper citation: Addgene The Nonprofit Plasmid Repository 2014. Restriction Digest of Plasmid DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.cd2s8d Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for how to perform DNA ligation. To see the full abstract and other resources, visit https://www.addgene.org/protocols/dna-ligation/.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. DNA Ligation. protocols.io https://dx.doi.org/10.17504/protocols.io.4g7gtzn Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: The following protocol is for making LB agar plates for the purpose of bacterial selection (500mL of LB agar makes about 25 LB agar plates). Please see the Addgene website for additional details.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Pouring LB Agar Plates. protocols.io https://dx.doi.org/10.17504/protocols.io.5icg4aw Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol goes through AAV titration by qPCR using SYBR Green Technology. To see the full abstract and additional resources, visit the Addgene protocol page.Sample DataReferencesAurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I, Nitschko H, Busch U, Sing A, Ehrhardt A, Baiker A. Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods. 2012 Feb;23(1):18-28.PMID: 22428977
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. AAV Titration by qPCR Using SYBR Green Technology. protocols.io https://dx.doi.org/10.17504/protocols.io.47kgzkw Copy
Authors: Ken Christensen, Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for agarose gel electrophoresis. To see the full abstract and additional resources, visit the Addgene protocol page.
Proper citation: Ken Christensen, Addgene The Nonprofit Plasmid Repository 2020. Agarose Gel Electrophoresis-Chem 584 . protocols.io https://dx.doi.org/10.17504/protocols.io.bjvnkn5e Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for restriction digest of plasmid DNA. To see the full abstract and additional resources, please visit http://www.addgene.org/plasmid_protocols/restriction_digest/.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Restriction Digest of Plasmid DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.63shgne Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for generating stable cell lines with lentivirus. To see the full abstract and additional resources, please visit the Addgene protocol page.Sample Data
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Generating Stable Cell Lines with Lentivirus. protocols.io https://dx.doi.org/10.17504/protocols.io.4xtgxnn Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes plasmid cloning by Polymerase Chain Reaction (PCR). To see the full abstract and additional resources, please visit the Addgene protocol page.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Plasmid Cloning by PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.43rgym6 Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes ddPCR titration of Lentivirus vectors. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/lentivirus-ddpcr-titration/.This protocol was modified from the publication Wang Y, Bergelson S, Feschenko M, 2018. Sample Data: When analyzing data there should be a clear distinction between negative droplets (black) and positive droplets (blue/green).The concentration of RRE positive droplets in the untransduced control should be close to zero (A01).In this protocol, the lentiviral particles are serially diluted and used to transduce HEK293T cells. Genomic DNA is extracted from the target cells and assayed for integrated copies of RRE. Since the samples that are assayed are diluted 2-fold serially, the concentration of RRE positive droplets should decrease by a factor of 2 across the dilutions. RPP30 copies should be relatively constant across samples.In the RRE example below, 2-fold serial dilutions of a sample were loaded in wells B01-H01.As shown in the image and table below, the concentration of RRE positive droplets increases by a factor of ~2 as you progress from the higher dilutions to the lower dilutions (blue).The concentration of RPP30 positive droplets stays relatively even across samples (green).To increase the accuracy of the titer, calculate and average of several dilutions.
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. ddPCR Titration of Lentivirus Vectors. protocols.io https://dx.doi.org/10.17504/protocols.io.be7ijhke Copy
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.
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. ddPCR titration of AAV vectors. protocols.io https://dx.doi.org/10.17504/protocols.io.bqifmubn Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is used for AAV production in HEK293T cells. To see the full abstract and additional resources, visit the Addgene protocol page.Sample Data
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. AAV Production in HEK293T Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bawpifdn Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for running a diagnostic restriction digest. To see the full abstract and other resources, visit the Addgene protocol page.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Diagnostic Restriction Digest. protocols.io https://dx.doi.org/10.17504/protocols.io.4gvgtw6 Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for Lentivirus production. To see the full abstract and additional resources, visit the Addgene protocol page.Sample Data
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Lentivirus Production. protocols.io https://dx.doi.org/10.17504/protocols.io.4xwgxpe Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes Ligation Independent Cloning (LIC). To see the full abstract and additional resources, please visit the Addgene protocol page.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Ligation Independent Cloning. protocols.io https://dx.doi.org/10.17504/protocols.io.bawjifcn Copy
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