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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 dx.doi.org/10.17504/protocols.io.47jgzkn 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 dx.doi.org/10.17504/protocols.io.bawrifd6 Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes how to design a primer. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/primer-design/.
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. How to Design a Primer. protocols.io dx.doi.org/10.17504/protocols.io.4r5gv86 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 dx.doi.org/10.17504/protocols.io.4xwgxpe Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes plasmid modification by annealed oligo cloning. To see the full abstract and additional resources, please visit the Addgene protocol page.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Plasmid Modification by Annealed Oligo Cloning. protocols.io dx.doi.org/10.17504/protocols.io.bawkifcw Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for DNA quantification. To see the full abstract and additional resources, please visit https://www.addgene.org/protocols/dna-quantification/.
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. DNA Quantification. protocols.io dx.doi.org/10.17504/protocols.io.4g8gtzw 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 dx.doi.org/10.17504/protocols.io.bawxiffn Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for isolating monoclonal cell population by limiting dilution. To see the full abstract and additional resources, please visit the Addgene protocol page.Sample Data1lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid #52962) and is described in Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Isolating a Monoclonal Cell Population by Limiting Dilution. protocols.io dx.doi.org/10.17504/protocols.io.4xvgxn6 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 dx.doi.org/10.17504/protocols.io.43rgym6 Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes plasmid modification by annealed oligo cloning. To see the full abstract and additional resources, please visit the Addgene protocol page.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Plasmid Modification by Annealed Oligo Cloning. protocols.io dx.doi.org/10.17504/protocols.io.47tgznn 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 dx.doi.org/10.17504/protocols.io.be7ijhke 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 dx.doi.org/10.17504/protocols.io.bjvnkn5e Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes Gibson Assembly cloning (Nat Methods 2009;6(5):343-5). To see the full abstract and additional resources, please visit the Addgene protocol page.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Gibson Assembly Cloning. protocols.io dx.doi.org/10.17504/protocols.io.43sgyne Copy
Authors: Joanne Kamens, Addgene The Nonprofit Plasmid Repository
Group: Re-Entering Labs Post COVID-19 Shutdown
Summary: Disclaimer: The SOP presented here has been designed by the Addgene nonprofit plasmid repository and is being shared outside of Addgene for informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that you do so at your own risk; you should ensure that any local guidance is also adhered to. None of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to from these web pages.
Proper citation: Joanne Kamens, Addgene The Nonprofit Plasmid Repository 2020. Addgene Covid-19, Back To Work Training. protocols.io dx.doi.org/10.17504/protocols.io.bhukj6uw Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: The following protocol is for bacterial transformation. To see the full abstract and additional resources, visit the Addgene protocol page.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Bacterial transformation. protocols.io dx.doi.org/10.17504/protocols.io.4f9gtr6 Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for isolating monoclonal cell population by limiting dilution. To see the full abstract and additional resources, please visit the Addgene protocol page.Sample Data1lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid #52962) and is described in Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Isolating a Monoclonal Cell Population by Limiting Dilution. protocols.io dx.doi.org/10.17504/protocols.io.bawwiffe 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 dx.doi.org/10.17504/protocols.io.5icg4aw 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 dx.doi.org/10.17504/protocols.io.bef8jbrw Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for recovering plasmid DNA from Bacterial Culture. To see the full abstract and other resources, visit https://www.addgene.org/protocols/purify-plasmid-dna/.
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. Recovering Plasmid DNA from Bacterial Culture. protocols.io dx.doi.org/10.17504/protocols.io.4gtgtwn 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 dx.doi.org/10.17504/protocols.io.47kgzkw Copy
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