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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 dx.doi.org/10.17504/protocols.io.cd2s8d Copy
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.bawqifdw 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.bawhifb6 Copy
Authors: Addgene The Nonprofit Plasmid Repository, Ken Christensen
Summary: This protocol describes how to streak and isolate (single colony) bacteria on an LB agar plate. To see the full abstract and additional resources, please visit https://www.addgene.org/protocols/streak-plate/.Sample Data
Proper citation: Addgene The Nonprofit Plasmid Repository, Ken Christensen 2020. Streaking and Isolating Bacteria on an LB Agar Plate - CHEM 584. protocols.io dx.doi.org/10.17504/protocols.io.bj5mkq46 Copy
Authors: Joanne Kamens, Addgene The Nonprofit Plasmid Repository
Group: Re-Entering Labs Post COVID-19 Shutdown
Proper citation: Joanne Kamens, Addgene The Nonprofit Plasmid Repository 2020. COVID-19 Addgene Operating Procedures. protocols.io dx.doi.org/10.17504/protocols.io.bhvej63e 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.bawgifbw 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). For the full abstract and additional resources, please visit https://www.addgene.org/plasmid_protocols/bacterial_plates/
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Pouring LB Agar Plates. protocols.io dx.doi.org/10.17504/protocols.io.63khgkw Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for performing Polymerase Chain Reaction (PCR). To see the full abstract and additional resources, visit https://www.addgene.org/protocols/pcr/.Basic PCR ProgramInitial Denaturation for at : This initiation step heats the double stranded DNA template strand to the point where the strands start denaturing and the hydrogen bonds are broken between the nucleotide base pairs.Denature at : Continued denaturation of double stranded DNA.Anneal primers for at : The forward and reverse primers are stable within this temperature range to anneal to each of the single stranded DNA template strands. The DNA polymerase is also stable enough to now bind to the primer DNA sequence.Extend DNA for at : The Taq polymerase has an optimal temperature around - so this step enables the DNA polymerase to synthesize and elongate the new target DNA strand accurately and rapidly.Repeat steps 2-5, 25-30 times.Final Extension for at : A final extension to fill-in any protruding ends of the newly synthesized strands.
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. Polymerase Chain Reaction (PCR). protocols.io dx.doi.org/10.17504/protocols.io.betyjepw 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.bjq3kmyn Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for purifying DNA from an agarose gel. To see the full abstract and additional resources, please visit https://www.addgene.org/protocols/gel-purification/.
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. Purifying DNA from an Agarose Gel. protocols.io dx.doi.org/10.17504/protocols.io.4wigxce Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for sequence analysis of a plasmid. To see the full abstract and additional resources, please visit https://www.addgene.org/protocols/sequence-analysis/.Sample dataA good sequencing reaction will produce between 300-900 base pairs of useable sequence. You should receive your sequencing results as a trace file (.ab1) which graphically depicts the sequence as a series of colored peaks corresponding to one of the four nucleotide bases. This is an example of a trace file from a high-quality portion of a sequencing reaction:Sequence near the beginning or end of a sequencing reaction is often unreliable. Although your sequencing results may indicate bases at specific locations, by looking at the trace file, you will see that these base calls are unreliable. This is an example of a trace file from a portion of a sequencing reaction with high background:
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. Sequence Analysis of a Plasmid. protocols.io dx.doi.org/10.17504/protocols.io.43ngyme Copy
Authors: 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: Addgene The Nonprofit Plasmid Repository 2019. Agarose Gel Electrophoresis. protocols.io dx.doi.org/10.17504/protocols.io.3ssgnee Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for over-agar antibiotic plating. To see the full abstract and additional resources, visit https://www.addgene.org/protocols/over-agar-antibiotic-plating/Sample Data: Selection of E.coli on LB-agar using different concentrations of carbenicillin plated over-agar.Control Plate with No CarbenicillinPlate shows a lawn of E. coli and no selection.150 µL of 0.1 mg/mL Carbenicillin plated over-agarPlate shows a lawn of E. coli and no apparent selection.150 µL of 1 mg/mL Carbenicillin plated over-agarPlate shows several individual colonies and effective selection.150 µL of 2 mg/mL Carbenicillin plated over-agarPlate shows less individual colonies than the 1 mg/mL plate and effective selection.150 µL of 4 mg/mL Carbenicillin plated over-agarPlate shows several individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection.Selection Curve of Transformed E. coli after Over-Agar Plating of Carbenicillin. The above graph displays the stock concentration of Carbenicillin stock used (150 µL per plate). Please note we have found that there is generally a broad range of antibiotic concentrations that will work for this assay, and the above result represents a single experiment. For publishable data, the experiment would need to be repeated to account for variability.
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. Over-Agar Antibiotic Plating. protocols.io dx.doi.org/10.17504/protocols.io.4r6gv9e Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This is the protocol accompanying the "pLKO.1 – TRC Cloning Vector". For information about the PLKO.1-TRC cloning vector and tips on designing shRNA oligos for pLKO.1 see Addgene's website: http://www.addgene.org/tools/protocols/plko/
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Cloning shRNA Oligos into pLKO.1. protocols.io dx.doi.org/10.17504/protocols.io.bawmifc6 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 dx.doi.org/10.17504/protocols.io.4gvgtw6 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 dx.doi.org/10.17504/protocols.io.43tgynn 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 dx.doi.org/10.17504/protocols.io.bawjifcn Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes how to streak and isolate (single colony) bacteria on an LB agar plate. To see the full abstract and additional resources, please visit https://www.addgene.org/protocols/streak-plate/.Sample Data
Proper citation: Addgene The Nonprofit Plasmid Repository 2020. Streaking and Isolating Bacteria on an LB Agar Plate. protocols.io dx.doi.org/10.17504/protocols.io.43pgymn 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.bawiifce 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 dx.doi.org/10.17504/protocols.io.63shgne Copy
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